2,4-dinitrophenol formulations and methods using same

ABSTRACT

The present invention includes a low dose and sustained release formulation of 2,4-dinitrophenol (DNP). The compositions of the invention are useful for preventing or treating a disease or disorder, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, insulin resistance and/or diabetes, in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, International Application No.PCT/US2014/053406, filed Aug. 29, 2014, and published under PCT Article21(2) in English, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 61/872,294, filed Aug. 30, 2013 and U.S.Provisional Application No. 61/919,003, filed Dec. 20, 2013, all ofwhich applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DK085638, DK040936and DK049230 awarded by National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Non-alcoholic fatty liver disease (NAFLD) is a key factor in thepathogenesis of type 2 diabetes (T2D) and affects one in three Americans(Shulman, 2000, J. Clin. Invest. 106:171-176; Petersen, et al., 2005,Diabetes 54:603-608; Samuel & Shulman, 2012, Cell 148:852-857; Boyle, etal., 2010, Popul. Health. Metr. 8:29). NAFLD is also a key predisposingfactor for the development of non-alcoholic steatohepatitis (NASH),cirrhosis and hepatocellular carcinoma. Further, NAFLD-induced NASH maysoon surpass hepatitis C and alcoholic cirrhosis as the most commonindication for liver transplantation in the USA (Sanyal, et al., 2010,Oncologist 15 Suppl. 4:14-22; Stickel & Hellerbrand, 2010, Gut59:1303-1307; Barry, et al., 2010, J. Hepatol. 56:1384-1391; White, etal., 2012, Clin. Gastroenterol. Hepatol. 10:1342-1359). Thus, new andeffective therapies for treatment of NAFLD are urgently needed.

One of the best characterized mitochondrial uncoupling agents is2,4-dinitrophenol (DNP), a protonophore that shuttles protons across themitochondrial membrane, dissipating the mitochondrial proton gradientand promoting heat dissipation of the energy derived from mitochondrialsubstrate oxidation. DNP was extensively used as a weight loss remedy inthe 1930s but taken off the market by the U.S. Food and DrugAdministration in 1938 due to the occurrence of fatal hyperthermia(Tainter, et al., 1934, Am. J. Public Health Nations Health24:1045-1053).

There is a need in the art for compositions useful for treating NAFLDand other diseases and disorders. The present invention addresses thisunmet need.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of preventing or treating a disease ordisorder in a subject in need thereof. The invention further provides amethod of increasing energy expenditure in a subject in need thereof.The invention further provides a therapeutically effective amount of apharmaceutically composition comprising a compound selected from thegroup consisting of 2,4-dinitrophenol (DNP), a salt thereof, a solvatethereof, and any combinations thereof, wherein the compound is in asustained release formulation.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound selected from the group consisting ofDNP, a salt thereof, a solvate thereof, and any combinations thereof.

In certain embodiments, the disease or disorder is at least one selectedfrom the group consisting of non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), hepatic steatosis, type 2 diabetes(T2D), acquired lipodystrophy, inherited lipodystrophy, partiallipodystrophy, hypertriglyceridemia, obesity, metabolic syndrome, Rett'ssyndrome, metabolic syndrome associated with aging, metabolic diseasesassociated with increased reactive oxygen species (ROS), Friedreich'sataxia, insulin resistance, hepatic fibrosis, liver cirrhosis andhepatocellular carcinoma.

In certain embodiments, the subject is afflicted with at least onedisease or disorder selected from the group consisting of NAFLD, NASH,hepatic steatosis, T2D, acquired lipodystrophy, inherited lipodystrophy,partial lipodystrophy, hypertriglyceridemia, obesity, metabolicsyndrome, Rett's syndrome, metabolic syndrome associated with aging,metabolic diseases associated with increased ROS, Friedreich's ataxia,insulin resistance, hepatic fibrosis, liver cirrhosis and hepatocellularcarcinoma.

In certain embodiments, the therapeutically effective dose of thecompound ranges from about 1 mg/kg/day to about 10 mg/kg/day. In otherembodiments, administration of the composition affords a steady stateplasma concentration of the compound ranging from about 0.05 μM to about200 μM in the subject. In yet other embodiments, administration of thecomposition affords a steady state plasma concentration of the compoundranging from about 0.5 μM to about 50 μM in the subject. In yet otherembodiments, administration of the composition affords a steady stateplasma concentration of the compound ranging from about 3 μM to about 5μM in the subject.

In certain embodiments, the steady state plasma concentration of thecompound in the subject is about 50 to about 100 times lower than thetoxic concentration of the compound in the subject. In otherembodiments, administration of the composition affords therapeuticallyeffective levels of the compound in the subject for a period of timeranging from about 12 hours to about 24 hours.

In certain embodiments, the composition is administered once, twice orthree times a day to the subject. In other embodiments, administrationof the composition does not cause significant systemic toxicity orsignificant increase in body temperature in the subject. In yet otherembodiments, the significant systemic toxicity is indicated by increasein levels of liver enzymes, blood urea nitrogen or creatinine ascompared to the corresponding levels in the subject in the absence ofadministration of the composition. In yet other embodiments, thecomposition is formulated for oral administration.

In certain embodiments, the subject is further administered at least oneadditional therapeutic agent. In other embodiments, the composition andthe at least one additional therapeutic agent are co-administered to thesubject. In yet other embodiments, the composition and the at least oneadditional therapeutic agent are co-formulated.

In certain embodiments, the subject is a mammal. In other embodiments,the mammal is human.

In certain embodiments, administration of the amount of the compositionto a subject affords a steady state plasma concentration of the compoundranging from about 0.05 μM to about 200 μM in the subject. In otherembodiments, administration of the amount of the composition to asubject affords a steady state plasma concentration of the compoundranging from about 0.5 μM to about 50 μM in the subject. In yet otherembodiments, administration of the amount of the composition to asubject affords a steady state plasma concentration of the compoundranging from about 3 μM to about 5 μM in the subject. In yet otherembodiments, the steady state plasma concentration of the compound inthe subject is about 50 to about 100 times lower than the toxicconcentration of the compound in the subject. In yet other embodiments,administration of the amount of the composition affords therapeuticallyeffective levels of the compound in the subject for a period of timeranging from about 12 hours to about 24 hours. In yet other embodiments,the amount of the composition is administered once, twice or three timesa day to the subject.

In certain embodiments, administration of the amount of the compositiondoes not cause significant systemic toxicity or significant increase inbody temperature in the subject. In other embodiments, the significantsystemic toxicity is indicated by increase in levels of liver enzymes,blood urea nitrogen or creatinine, as compared to the correspondinglevels in the subject in the absence of administration of thecomposition. In yet other embodiments, the composition is formulated fororal administration. In yet other embodiments, the composition furthercomprises at least one additional therapeutic agent.

In certain embodiments, the compound in the composition is coated with acoating comprising at least one selected from the group consisting ofhydroxypropylcellulose and ethylcellulose. In other embodiments, thecoating further comprises at least one selected from the groupconsisting of talc and dibutyl sebacate. In yet other embodiments, thecompound is in a bead or sphere form. In yet other embodiments, the beador sphere comprising the compound further comprises at least oneselected from the group consisting of mannitol, microcrystallinecellulose and hydroxypropylmethyl cellulose.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings specific embodiments. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities of the embodiments shown in the drawings.

FIG. 1 illustrates the structure of 2,4-dinitrophenol (DNP).

FIG. 2, comprising FIGS. 2A-2H, illustrates the finding that low-doseintragastric infusion of DNP reduces plasma glucose, triglyceride andinsulin concentrations, as well as hepatic steatosis and whole bodyinsulin resistance, in a rat model of NAFLD and whole body insulinresistance. FIG. 2A is a graph illustrating plasma, liver, andquadriceps DNP concentrations. FIG. 2B is a graph illustrating livertriglyceride content. FIG. 2C is a graph illustrating muscletriglyceride content. FIG. 2D is a graph illustrating liverdiacylglycerol content. FIG. 2E is a graph illustrating musclediacylglycerol content. FIG. 2F is a graph illustrating fasting plasmaglucose concentrations. FIG. 2G is a graph illustrating fasting plasmatriglyceride concentrations. FIG. 2H is a graph illustrating fastingplasma insulin concentrations. n=3-4 per group.

FIG. 3 is a graph illustrating plasma DNP concentrations after feedingrats extended release DNP (ERDNP) (1 mg/kg) in peanut butter at time 0.Treatment with ERDNP achieved a sustained plasma concentration of DNPbetween 3-8 μM for at least 12 hours.

FIG. 4, comprising FIGS. 4A-4D, illustrates the finding that ERDNP has a500-fold wider ratio of effective to safe dose than DNP. FIGS. 4A-4B:Rectal temperature of rats treated acutely with DNP or ERDNP. FIGS.4C-4D: Liver triacylglycerol concentration in high fat fed rats treatedfor 5 days with DNP or ERDNP. *P<0.05, **P<0.01, ***P<0.001 versusvehicle-treated group. n=3 per dose, per group.

FIG. 5, comprising FIGS. 5A-5F, illustrates the finding that ERDNP isbetter tolerated than DNP because it results in lower plasma and tissueDNP concentrations. FIG. 5A: Plasma concentrations after a dose of DNP(25 mg/kg, toxic dose; black circles) or ERDNP (1 mg/kg; red squares).FIG. 5B: Tissue DNP concentrations 1 hour after treatment with 25 mg/kgDNP. FIG. 5C-5D: Tissue DNP concentrations after one or five daily dosesof 1 mg/kg ERDNP. FIG. 5E: Tissue DNP concentrations after 6 weeks ofdaily treatment with 1 mg/kg ERDNP. FIG. 5F: Tissue DNP concentrations24 hours after ERDNP treatment. In all panels, n=3 per group.

FIG. 6, comprising FIGS. 6A-6G, illustrates the finding that ERDNP (1mg/kg per day for 5 days) reverses NAFLD and improves glucose toleranceand insulin sensitivity in high fat fed Sprague-Dawley rats. FIGS.6A-6C: Fasting plasma glucose, triglyceride and insulin concentrations.FIG. 6D: Plasma cholesterol concentrations. FIGS. 6E-6F: Plasma glucoseand insulin concentrations during an IP glucose tolerance test. FIG. 6G:Fasting plasma non-esterified fatty acids concentrations. *P<0.05,**P<0.01, ***P<0.001. In all panels, n=6-8 per group.

FIG. 7, comprising FIGS. 7A-7D, illustrates the finding that ERDNP (1mg/kg per day for 5 days) improves insulin sensitivity in high fat fedrats. FIG. 7A: Glucose infusion rate to maintain euglycemia in ahyperinsulinemic-euglycemic clamp. FIG. 7B: Insulin-stimulated glucoseuptake in quadriceps muscle. FIG. 7C: Basal (solid bars) andinsulin-stimulated (dashed bars) hepatic glucose production. FIG. 7D:Insulin-stimulated suppression of hepatic glucose production. In allpanels, n=6-8 per group.

FIG. 8, comprising FIGS. 8A-8F, illustrates the finding that ERDNP (1mg/kg per day for 5 days) reduces hepatic gluconeogenesis in high fatfed rats. FIGS. 8A-8B: Liver and quadriceps triacylglycerol. FIG. 8C:Liver pyruvate carboxylase flux. FIG. 8D: Liver acetyl CoAconcentration. FIG. 8E: Hepatic tricarboxylic acid cycle (TCA) cycleflux supplied by carbons from fatty acid oxidation (solid bars) andthrough PDH flux (dashed bars). FIG. 8F: Hepatic triglyceride export.n=6 per group.

FIG. 9, comprising FIGS. 9A-9H, illustrates the finding that oral ERDNP(1 mg/kg per day for 14 days) reverses NAFLD and improves glucosetolerance in Zucker Diabetic Fatty rats. FIG. 9A: Random plasma glucoseconcentrations in vehicle-treated (black circles) and ERDNP-treated rats(red squares). FIGS. 9B-9D: Fasting plasma glucose, triglyceride andinsulin concentrations. FIGS. 9E-9F: Glucose and insulin concentrationsduring an intraperitoneal glucose tolerance test. FIGS. 9G-9H: ALT andAST concentrations. FIG. 9I: Liver histology (hematoxylin & eosinstain). In all panels, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.n=6-7 per group.

FIG. 10 is a graph illustrating fasting plasma glucose. Rats were fastedfor 6 hours. ERDNP oral dosing (1 mg/kg every 12 hours) for 5 daysresults in significant reductions in fasting plasma glucoseconcentrations in a high fat fed rat model of insulin resistance andNAFLD.

FIG. 11, comprising FIGS. 11A-11B, illustrates tissue TAG content aftertreatment with vehicle or ERDNP. FIG. 11A is a graph illustrating liverTAG content. FIG. 11B is a graph illustrating quadriceps TAG content.Rats were fasted for 6 hours. ERDNP oral dosing (1 mg/kg every 12 hours)for 5 days resulted in significant reductions in liver triglycerideconcentrations and a strong tendency for reductions in muscletriglyceride content in a high fat fed rat model of insulin resistanceand NAFLD.

FIG. 12 is a graph illustrating how single day dosing of extendedrelease DNP (ERDNP) for 5 days shows a strong tendency to reduce plasmainsulin concentrations. Rats were treated once daily with 1 mg/kg ERDNPor vehicle for 5 days. Prior to sacrifice, rats were fasted for 6 hours.

FIG. 13 is a graph illustrating how single day dosing of extendedrelease DNP (ERDNP) for 5 days results in significant reductions inHOMA-IR. This reduction in HOMA-IR demonstrated that single day dosingof ERDNP reduced whole body insulin resistance in a high fat rodentmodel of insulin resistance.

FIG. 14, comprising FIGS. 14A-14L, illustrates the finding that chronic,intragastric DNP infusion (2 mg/kg per day) safely reverses NAFLD inrats. FIGS. 14A-14B: Liver and quadriceps diacylglycerol. FIGS. 14C-14D:Liver and quadriceps DAG species. FIGS. 14E-14F: Liver and quadricepsceramides. FIGS. 14G-14J: ALT, AST, BUN, and creatinine concentrations.FIG. 14K: Body weight before and after treatment. FIG. 14L: Dailycaloric intake during the treatment period. n=4 per group.

FIG. 15, comprising FIGS. 15A-15H, illustrates the finding that ERDNPhas a 500-fold wider ratio of effective to safe dose than DNP. FIGS.15A-15B: ALT in rats treated with varying doses of DNP or ERDNP for 5days. FIGS. 15C-15D: AST in rats treated with varying doses of DNP orERDNP for 5 days. FIGS. 15E-15F: BUN in rats treated with varying dosesof DNP or ERDNP for 5 days. FIGS. 15G-15H: Creatinine in two-week highfat fed rats treated with varying doses of DNP for five days. n=3 perdose, per group.

FIG. 16, comprising FIGS. 16A-16H, illustrates the finding that sixweeks of ERDNP treatment (1 mg/kg per day) is well tolerated in rats.FIGS. 16A-16D: ALT, AST, BUN, and creatinine. FIGS. 16E-16F:Representative images of liver and kidney, respectively, stained withhematoxylin and eosin. FIG. 16G: Rectal temperature. FIG. 16H: TissueDNP concentrations 8 hours after the last dose of ERDNP. In all panels,n=3-6 per group.

FIG. 17, comprising FIGS. 17A-17K, illustrates the finding that ERDNP (1mg/kg per day for 5 days) reverses NAFLD and improves glucose toleranceand insulin sensitivity in high fat fed Sprague-Dawley rats. FIG. 17A:Body weight at the end of the treatment period. FIG. 17B: Non-esterifiedfree fatty acid concentration. FIG. 17C: Western blots. FIGS. 17D-17F:Pyruvate carboxylase, glucose-6-phosphatase, andfructose-1,6-bisphosphatase protein. FIGS. 17G-17H: Plasma glucose andinsulin area under the curve in an IP glucose tolerance test. In allpanels, n=6-8 per group. FIG. 17I: Ratio of fat oxidation to TCA cycleflux. Black bars, vehicle; red/gray bars, ERDNP. FIG. 17J: White adiposetissue weight. FIG. 17K: Plasma cholesterol. Black bars, vehicle;red/gray bars, ERDNP.

FIG. 18, comprising FIGS. 18A-18Z, illustrates the finding that ERDNP (1mg/kg per day for 5 days) ameliorates NAFLD improves insulin sensitivityin high fat fed rats. FIG. 18A: Plasma insulin concentrations at the endof a 120 min hyperinsulinemic-euglycemic clamp. FIG. 18B: Plasma glucoseconcentrations throughout the clamp. FIG. 18C: Glucose infusion rateduring the clamp. FIGS. 18D-18E: Liver and quadriceps DAG species. FIGS.18F-18G: Liver PKCε and quadriceps PKCθ translocation. FIGS. 18H-18J:Liver acylcarnitine concentrations. FIGS. 18K-18M: Quadricepsacylcarnitine concentrations. FIGS. 18N-18O: Liver and quadricepsceramide concentrations. FIG. 18P: Liver glycogen content. FIG. 18Q:Plasma inflammatory cytokine concentrations. n=3 per group. FIGS.18R-18S: Plasma adiponectin and FGF-21 concentrations. FIG. 18T:Insulin-stimulated glucose uptake in quadriceps. FIGS. 18U-18V: Liverand quadriceps DAG content. FIG. 18X: Intrascapular brown adipose tissuemass. FIG. 18Y: BAT UCP1 mRNA expression normalized to actin. FIG. 18Z:Insulin-stimulated glucose uptake in brown adipose tissue. Unlessotherwise stated, n=6-8 per group.

FIG. 19, comprising FIGS. 19A-19L, illustrates the finding that ERDNP (1mg/kg per day for 14 days) reverses NAFLD and improves glucose tolerancein Zucker Diabetic Fatty rats. FIG. 19A: Body weight before and aftertreatment with vehicle (black bars) or ERDNP (red bars). FIGS. 19B-19C:Glucose and insulin area under the curve in the IP glucose tolerancetest. FIGS. 19D-19E: Liver and quadriceps TAG concentrations. FIGS.19F-19I: ALT, AST, BUN, and creatinine concentrations. FIGS. 19J-19K:Liver acetyl and malonyl CoA concentrations. FIG. 19L: Hepatic acetylCoA concentration. In all panels, n=6-7 per group.

FIG. 20, comprising FIGS. 20A-20L, illustrates the finding that oralERDNP (1 mg/kg per day) ameliorates NASH and improves liver syntheticfunction in methionine/choline deficient rats. FIG. 20A: Livertriglyceride content. FIGS. 20B-20C: Plasma AST and ALT concentrations.FIG. 20D: Liver inflammatory cytokine concentrations, normalized tototal protein. n=4 per group. FIG. 20E: Liver histology. FIG. 20F:Fibrosis score. FIG. 20G: Liver collagen mRNA expression. FIG. 20H:Liver smooth muscle actin protein. FIG. 20I: Hepatic hydroxyprolinecontent. FIGS. 20J-20K: Liver caspase 3 and caspase 9 protein. FIG. 20L:Plasma albumin concentrations. Unless otherwise specified, n=6-8 pergroup.

FIG. 21, comprising FIGS. 21-21L, illustrates the finding that ERDNP isbetter tolerated than DNP because it results in lower plasma and tissueDNP concentrations. FIGS. 21A-21B: Plasma DNP concentrations after 1mg/kg (FIG. 21A) or 25 mg/kg (FIG. 21B) DNP at time 0. n=3-4. FIGS.21C-21D: Plasma DNP concentrations after 1 mg/kg (FIG. 21C) or 25 mg/kg(FIG. 21D) ERDNP at time 0. n=6-9. FIG. 21E: 24 hour area under thecurve of plasma DNP concentrations after treatment with 1 mg/kg oral DNP(n=4) or ERDNP (n=9). FIG. 21F: Correlation between rectal temperatureand plasma DNP concentration in rats treated with 10-50 mg/kg DNP. FIG.21G: Tissue DNP concentrations 1 hour after treatment with 25 mg/kg DNP.n=3. FIG. 21H: Tissue DNP concentrations 8 hours after one dose of 1mg/kg orally administered ERDNP. n=3. FIG. 21I: Plasma:tissue DNP ratioat various time points following 1 mg/kg oral ERDNP. n=3. FIG. 21J:Tissue DNP concentrations 8 hours after the last of five daily 1 mg/kgERDNP doses. n=3. FIG. 21K: Linear correlations between oral ERDNP doseand tissue DNP concentrations measured 8 hours after dosing. n=3. FIG.21L: Plasma DNP concentrations after one dose and the last of five daily1 mg/kg ERDNP doses. Data for treatment-naïve rats are copied from FIG.21C. n=4 for rats treated chronically.

FIG. 22, comprising FIGS. 22A-22F, illustrates the finding that twoweeks of daily ERDNP treatment (1 mg/kg) prevented NAFLD and insulinresistance in rats concurrently fed high fat diet. FIGS. 22A-22C:Fasting plasma glucose, NEFA and insulin concentrations. FIGS. 22D-22F:Liver, plasma, and quadriceps triglyceride content. n=8 per group.

FIG. 23, comprising FIGS. 23A-23H, illustrates the finding that ERDNPdid not cause any physiologically significant difference in anyparameter measured in metabolic cage analysis in mice. FIG. 23A: Oxygenconsumption (V_(O2)). FIG. 23B: Carbon dioxide production (V_(CO2)).FIG. 23C: Respiratory quotient. FIG. 23D: Activity over the course ofthe day. FIG. 23E: Energy expenditure throughout the day. FIG. 23F:Total daily water drinking. FIG. 23G: Total daily food intake. FIG. 23H:Food intake over the course of the day. *P<0.05, **P<0.01. n=8 pergroup.

FIG. 24, comprising FIGS. 24A-24E, illustrates the finding that sixweeks of daily ERDNP treatment (1 mg/kg per day) ameliorates NASH andimproves liver synthetic function in methionine/choline deficient rats.FIG. 24A: Liver inflammatory cytokine protein content, normalized tototal protein. FIG. 24B: Hepatic CD69 protein. FIG. 24C: Livers stainedfor TUNEL positive cells (brown stain). FIG. 24D: Fasting plasma glucoseconcentrations. FIG. 24E: Liver glycogen content. n=6-8 per group.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the unexpected discovery that a low, sustaineddose of 2,4-dinitrophenol (DNP), or a salt or solvate thereof, or anycombinations thereof, provides reductions in hepatic steatosis, improvedinsulin sensitivity, improved glucose tolerance, reduced blood glucoseand/or reversal in liver inflammation, without causing hyperthermia andsystemic toxicities.

In certain embodiments, the compositions of the invention are useful intreating or preventing at least one disease or disorder selected fromthe group consisting of non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), hepatic steatosis, type 2 diabetes(T2D), acquired lipodystrophy, (inherited) lipodystrophy, partiallipodystrophy, hypertriglyceridemia, obesity, metabolic syndrome, Rett'ssyndrome, metabolic syndrome associated with aging, metabolic diseasesassociated with increased reactive oxygen species (ROS), Friedreich'sataxia, insulin resistance, hepatic fibrosis, liver cirrhosis andhepatocellular carcinoma. In other embodiments, the compositions of theinvention are useful in managing, maintaining, and/or preventing theincrease of, the weight of a subject. In yet other embodiments, thecompositions of the invention are useful in reducing the weight of asubject.

In one aspect, the studies described herein demonstrate that thesystemic toxicities of a mitochondrial protonophore (such as DNP) may bedissociated from its ability to promote hepatic mitochondrial uncouplingand increase hepatic fat oxidation by altering its pharmacokinetics. Thefindings described herein should not be construed to be limited toextended release formulations of 2,4-DNP, but rather are applicable toextended release formulations of any known and useful mitocondrialprotonophore or analogue/derivative thereof, such as but not limited toany isomers and/or analogues of 2,4-DNP (e.g., beta-2,4-DNP and2,6-dinitrophenol), carbonyl cyanide m-chlorophenyl hydrazine (CCCP) andcarbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP).

As described herein, the safety and efficacy of a novel extended releaseoral formulation of DNP (ERDNP) on hepatic steatosis, insulin resistanceand diabetes was studied in rat models of NAFLD and T2D. Such modelsachieved sustained plasma concentrations of DNP (1 to 10 μM over 24hours), which are 50-100 fold lower than the toxic threshold for DNP. Ina non-limiting embodiment, low concentrations of DNP (1-5 μM) were foundto promote subtle increases in hepatic mitochondrial uncoupling whileremaining well below the toxic plasma threshold of DNP (˜400 μM).

ERDNP was found to have a therapeutic index that was 500-fold greaterthan DNP. Chronic ERDNP treatment reduced hypertriglyceridemia, hepaticsteatosis, insulin resistance and diabetes in rat models of NAFLD andT2D. Further, ERDNP normalized plasma transaminase concentrations,ameliorated liver fibrosis, and improved hepatic protein syntheticfunction as reflected by an increase in plasma albumin concentrations ina methionine/choline deficient rat model of NASH. Further, chronic ERDNPwas well tolerated and not associated systemic toxicities. These dataindicates that ERDNP may be used in treating related epidemics of T2D,NASH, hepatic fibrosis and metabolic syndrome.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “abnormal,” when used herein in the context of organisms,tissues, cells or components thereof, refers to those organisms,tissues, cells or components thereof that differ in at least oneobservable or detectable characteristic (e.g., age, treatment, time ofday, etc.) from those organisms, tissues, cells or components thereofthat display the “normal” (expected) respective characteristic.Characteristics that are normal or expected for one cell or tissue typemight be abnormal for a different cell or tissue type.

As used herein, the term “about” when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

A disease or disorder is “alleviated” or “treated” if the severity of asymptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, is reduced.

As used herein, the term “ALT” refers to alanine aminotransferase.

As used herein, the term “AST” refers to aspartate aminotransferase.

As used herein, the term “BUN” refers to blood urea nitrogen.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient.Multiple techniques of administering a compound exist in the artincluding, but not limited to, intravenous, oral, aerosol, inhalational,rectal, vaginal, transdermal, intranasal, buccal, sublingual,parenteral, intrathecal, intragastrical, ophthalmic, pulmonary andtopical administration.

A used herein, the term “CRMP” refers to controlled-release oralformulation of a mitochondrial protonophore. As used herein, the terms“ERDNP” and “CRMP” are used interchangeably.

As used herein, the term “DAG” refers to diacylglycerol.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the terms “DNP” and “2,4-DNP” refer to2,4-dinitrophenol, or a salt or solvate thereof, or any combinationsthereof (FIG. 1).

An “effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “efficacy” refers to the maximal effect(E_(max)) achieved within an assay.

As used herein, the term “ERDNP” refers to extended release2,4-dinitrophenol, or a salt or solvate thereof, or any combinationsthereof.

As used herein, the term “LC/MS/MS” refers to liquid chromatography/massspectrometry/mass spectrometry.

As used herein, the term “NAFLD” refers to non-alcoholic fatty liverdisease.

As used herein, the term “NMR” refers to nuclear magnetic resonance.

As used herein, the term “patient,” “individual” or “subject” refers toa human or a non-human mammal. Non-human mammals include, for example,livestock and pets, such as ovine, bovine, porcine, canine, feline andmurine mammals. Preferably, the patient, individual or subject is human.

As used herein, the term “PC” refers to pyruvate carboxylase.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids and bases, including inorganic acids,inorganic bases, organic acids, inorganic bases, solvates, hydrates, andclathrates thereof. Suitable pharmaceutically acceptable acid additionsalts may be prepared from an inorganic acid or from an organic acid.Examples of inorganic acids include sulfate, hydrogen sulfate,hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, andphosphoric acids (including hydrogen phosphate and dihydrogenphosphate). Appropriate organic acids may be selected from aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic andsulfonic classes of organic acids, examples of which include formic,acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic,phenylacetic, mandelic, embonic (pamoic), methanesulfonic,ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid. Suitable pharmaceutically acceptablebase addition salts of compounds of the invention include, for example,ammonium salts and metallic salts including alkali metal, alkaline earthmetal and transition metal salts such as, for example, calcium,magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptablebase addition salts also include organic salts made from basic aminessuch as, for example, N,N′-dibenzylethylene-diamine, chloroprocaine,choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine)and procaine. All of these salts may be prepared from the correspondingcompound by reacting, for example, the appropriate acid or base with thecompound.

As used herein, the term “PKCε” refers to protein kinase CE.

As used herein, the term “PKCθ” refers to protein kinase CO.

As used herein, the term “potency” refers to the dose needed to producehalf the maximal response (ED₅₀).

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

As used herein, the term “significant increase in body temperature” in asubject refers to a body temperature increase that is associated withdeleterious effects on the subject, not limited to illness, physicaldiscomfort or pain, coma and death. In one non-limiting embodiment, thesignificant increase in body temperature is an increase of about 0.5°C., about 1° C., about 1.5° C., about 2° C., about 2.5° C., about 3° C.,about 3.5° C., about 4° C., about 4.5° C., about 5° C., about 5.5° C.,about 6° C. or higher. In another non-limiting embodiment, thesignificant increase in body temperature lasts for about 5 min, about 15min, about 30 min, about 45 min, about 1 h, about 1.5 h, about 2 h,about 3 h, about 4 h, about 5 h, about 6 h, about 7 h, about 8 h, about9 h, about 10 h, about 12 h, about 14 h, about 16 h, about 18 h, about20 h, about 22 h, about 24 h or longer.

As used herein, the term “significant systemic toxicity” in a subjectrefers to systemic toxicity that is associated with deleterious effectson the subject, not limited to illness, physical discomfort or pain,coma and death. In one non-limiting embodiment, significant systemictoxicity is indicated by increase in levels of liver enzymes, blood ureanitrogen or creatinine as compared to the corresponding levels in thesubject in the absence of administration of the composition.

As used herein, the term “TAG” refers to triacylglycerol.

As used herein, the term “T2D” refers to type 2 diabetes.

As used herein, the term “TCA” refers to tricarboxylic acid cycle.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compounduseful within the invention (alone or in combination with anotherpharmaceutical agent), to a patient, or application or administration ofa therapeutic agent to an isolated tissue or cell line from a patient(e.g., for diagnosis or ex vivo applications), who has a disease ordisorder, a symptom of a disease or disorder or the potential to developa disease or disorder, with the purpose to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve or affect the disease ordisorder, the symptoms of the disease or disorder, or the potential todevelop the disease or disorder. Such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics.

As used herein, the term “VLDL” refers to very low-density lipoprotein.

As used herein, the term “WAT” refers to white adipose tissue.

As used herein, the term “ZDF” refers to Zucker Diabetic Fatty.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The invention relates to the unexpected discovery that a low, sustaineddose of DNP reduces hepatic steatosis, improves insulin sensitivity,improves glucose tolerance, reduces blood glucose and/or reverses liverinflammation in a subject, without causing hyperthermia and systemictoxicities in the subject. In certain embodiments, the subject is amammal. In yet other embodiments, the mammal is human.

The present invention relates in part to the unexpected discovery thatsignificant hepatic mitochondrial uncoupling may be achieved byadministering a very low dose continuous infusion of DNP (or any othermitochondrial protonophore) without any associated systemic toxicities,leading to increased hepatic fatty acid oxidation, decreased hepatic fatcontent, and reversal of hepatic and peripheral insulin resistance in arat model of non-alcoholic fatty liver disease (NAFLD). The toxicity ofDNP in prior art studies was due to the pharmacokinetics of oral DNPleading to peak plasma concentrations that were many fold greater thanwhat is required to achieve a therapeutic effect. In view of the factthat hyperthermia and related toxicities of DNP are on-target effectsrelated to systemic mitochondrial uncoupling, a slow release formulationof DNP is an effective and safe approach to promote the metabolism ofhepatic triglyceride, while avoiding hyperthermia and associatedsystemic toxicities that typically occur with mitochondrial uncouplingagents.

In one aspect, the present invention contemplates the use of anymitochondrial uncoupling agent within the compositions and methods ofthe invention. In certain embodiments, the mitochondrial uncouplingagent comprises DNP. In yet other embodiments, the mitochondrialuncoupling agent comprises CCCP, FCCP, 2,6-dinitrophenol, or any known2,4-DNP analog/derivative/isomer.

As demonstrated herein, sustained plasma and liver concentrations of DNPbetween 0.5 μM-50 μM, such as between 1-3 μM, which were achieved duringa continuous low dose intragastric infusion of DNP, reversed fatty liverand whole body insulin resistance in a well-established high fat ratmodel of NAFLD. These plasma DNP concentrations were ˜100 fold lowerthan the threshold where DNP toxicity is first detected. Based on theseresults, an extended release preparation of DNP was formulated thatresulted in low sustained plasma DNP concentrations over a 24-hourinterval, which in turn led to a 500-fold improvement in the ratio oftoxic to effective dose of ERDNP compared to DNP.

Chronic safety and efficacy studies of ERDNP treatment were performed,chronic daily ERDNP treatment was well tolerated in rats for up to 6weeks with no changes in behavior, body temperature, food intake,activity or body weight. Further, chronic ERDNP treatment was notassociated with any systemic or liver/renal toxicities, as indicated bya lack of elevations in liver enzymes, BUN, or creatinine or deleteriouschanges in liver or kidney histology.

In terms to efficacy, chronic ERDNP treatment resulted in a 50%reduction in liver TAG content. This reduction in hepatic fat contentcould be attributed to a 60% increase in liver mitochondrial V_(TCA)cycle flux, which was entirely due to a 70% increase in hepatic fatoxidation. This reduction in liver lipid content was associated withmarked reductions in fasting plasma glucose concentrations, basal ratesof hepatic glucose production and improvement in whole body insulinsensitivity. The reduction in basal rates of hepatic glucose productioncould be attributed to a 25% reduction in V_(PC) flux, which was mostlikely secondary to a 50% reduction in concentrations of acetyl CoA, akey allosteric regulator of PC activity. In contrast, there was noeffect of ERDNP treatment on hepatic PEP-CK, PC or G6Pase proteinexpression.

ERDNP improvement in whole body insulin sensitivity in turn could beattributed to an increase in both hepatic and muscle insulinresponsiveness, as reflected by a 2.5-fold increase in suppression ofhepatic glucose production during a hyperglycemic-euglycemic clamp and athree-fold increase in insulin-stimulated peripheral glucose uptake.This ERDNP induced increase in liver and muscle insulin sensitivitycould be attributed to >50% reductions in liver and muscle DAG content,as well as in PKCε and PKCθ activity in liver and muscle respectively.DAG-nPKC activation is implicated in causing liver and muscle insulinresistance in both humans and animal models of NAFLD. The observed ERDNPreduction in TAG/DAG content could be attributed at least in part to an80% reduction in hepatic VLDL export. Without wishing to be bound by anytheory, while ERDNP may promote mitochondrial uncoupling in skeletalmuscle, the fact that 20-fold higher DNP concentrations did not causesignificant mitochondrial uncoupling in the quadriceps makes thispossibility less likely.

The safety and efficacy of ERDNP were examined in ZDF rats, a model ofT2D associated with NASH. Two weeks of ERDNP treatment resulted in a 65%reduction in liver fat content and reversal of hepatic inflammation asreflected by normalization of plasma ALT and AST in these animals,highlighting a possible role for ERDNP in reversing NASH. In addition,ERDNP caused a 400 mg/dL reduction in fasting plasma glucoseconcentrations in ZDF rats, which could be attributed to markedimprovements in whole body insulin sensitivity as reflected by lowerplasma glucose and insulin concentrations during an intraperitionalglucose tolerance test. Consistent with the lack of systemic toxicitiesobserved in the other animal studies, chronic ERDNP treatment in ZDFrats was well tolerated and caused no alterations in behavior, bodytemperature, feeding behavior, body weight, or activity and wasunassociated with any changes in renal function.

In one aspect, the present studies indicate that, by altering thepharmacokinetics of DNP to promote a low sustained systemic release, thetherapeutic window of DNP is increased by more than 500-fold. ChronicERDNP administration reversed NAFLD/NASH, insulin resistance, and type 2diabetes in the rat without any systemic, hepatic or renal toxicity.Taken together, these data support the utility of ERDNP for thetreatment of the related epidemics of NAFLD/NASH, metabolic syndrome andtype 2 diabetes.

The compositions of the invention, which include therapeutic low dose,sustained release formulations of DNP, are useful in treating a diseaseor disorder, such as but not limited to non-alcoholic fatty liverdisease (NAFLD), non-alcoholic steatohepatitis (NASH), hepaticsteatosis, type 2 diabetes (T2D), acquired lipodystrophy, lipodystrophy(inherited), partial lipodystrophy, hypertriglyceridemia, obesity,metabolic syndrome, Rett's syndrome, metabolic syndrome associated withaging, metabolic diseases associated with increased reactive oxygenspecies (ROS), Friedreich's ataxia, insulin resistance, hepaticfibrosis, liver cirrhosis and hepatocellular carcinoma.

In certain embodiments, the compositions and methods of the presentinvention may be used to treat or prevent a disease or disorder such as,but not limited to, NAFLD, non-alcoholic steatohepatitis (NASH), hepaticsteatosis, acquired lipodystrophy, inherited lipodystrophy, partiallipodystrophy, insulin resistance, type 2 diabetes (T2D), obesity,hypertriglyceridemia, metabolic syndrome, metabolic syndrome associatedwith aging, metabolic diseases associated with increased reactive oxygenspecies (ROS), Friedreich's ataxia, hepatic fibrosis, liver cirrhosis,hepatocellular carcinoma, diseases in which free radical mediatedoxidative injury leads to tissue degeneration, and diseases in whichcells inappropriately undergo apoptosis, and include the treatment of awide number of diseases, including but not limited to auto-immunedisease, congenital muscular dystrophy, fatal infantile myopathy,“later-onset” myopathy, MELAS (mitochondrial encephalopathy, lacticacidosis, and stroke), MIDD (mitochondrial diabetes and deafness), MERRF(myoclonic epilepsy ragged red fiber syndrome), arthritis, NARP(Neuropathy; Ataxia; Retinitis Pigmentosa), MNGIE (Myopathy and externalophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy), LHON(Leber's; Hereditary; Optic; Neuropathy), Kearns-Sayre disease,Pearson's Syndrome, PEO (Progressive External Ophthalmoplegia), Wolframsyndrome, DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy,Deafness), ADPD (Alzheimer's disease; Parkinson's disease), AMFD(ataxia, myoclonus and deafness), CIPO (chronic intestinalpseudoobstruction; myopathy; opthalmoplegia), CPEO (chronic progressiveexternal opthalmoplegia), maternally inherited deafness,aminoglycoside-induced deafness, DEMCHO (dementia; chorea), DMDF(diabetes mellitus; deafness), exercise intolerance, ESOC (epilepsy;strokes; optic atrophy; congenitive decline), FBSN (familial bilateralstriatal necrosis), FICP (fatal infantile cardiomyopathy plus aMELAS-associated cardiomyopathy), GER (gastrointestinal reflux), LIMM(lethal infantile mitochondrial myopathy), LDYT (Leber's hereditaryoptic neuropathy and DYsTonia), MDM (myopathy; diabetes mellitus), MEPR(myoclonic epilepsy; psychomotor regression), MERME (MERRF/MELAS overlapdisease), MHCM (maternally inherited hypertrophic cardiomyopathy), MICM(maternally inherited cardiomyopathy), MILS (maternally inherited Leighsyndrome), mitochondrial encephalocardiomyopathy, mitochondrialencephalomyopathy, mitochondrial myopathy, MMC (maternal myopathy;cardio myopathy), multisystem mitochondrial disorder (myopathy;encephalopathy; blindness; hearing loss; peripheral neuropathy), NIDDM(non-insulin dependent diabetes mellitus), PEM (progressiveencephalopathy), PME (progressive myclonus epilepsy), Rett's syndrome,SIDS (sudden infant death syndrome, SNHL (sensorineural hearing loss),Leigh's Syndrome, dystonia, schizophrenia, and psoriasis.

In certain embodiments, the disease or disorder is NAFLD. In yet otherembodiments, the disease or disorder is non-alcoholic steatohepatitis(NASH). In yet other embodiments, the disease or disorder is hepaticsteatosis. In yet other embodiments, the disease or disorder is type 2diabetes. In yet other embodiments, the disease or disorder is acquiredlipodystrophy. In yet other embodiments, the disease or disorder isinherited lipodystrophy. In yet other embodiments, the disease ordisorder is partial lipodystrophy. In yet other embodiments, the diseaseor disorder is hypertriglyceridemia. In yet other embodiments, thedisease or disorder is obesity. In yet other embodiments, the disease ordisorder is metabolic syndrome. In yet other embodiments, the disease ordisorder is insulin resistance. In yet other embodiments, the disease ordisorder is Rett's syndrome. In yet other embodiments, the disease ordisorder is metabolic syndrome associated with aging. In yet otherembodiments, the disease or disorder is metabolic diseases associatedwith increased reactive oxygen species (ROS). In yet other embodiments,the disease or disorder is Friedreich's ataxia. In yet otherembodiments, the disease is hepatic fibrosis. In yet other embodiments,the disease is liver cirrhosis. In yet other embodiments, the disease ishepatocellular carcinoma.

Sustained Release and Controlled Release Formulations

In one aspect, the invention provides a formulation for a sustainedrelease of DNP. In certain embodiments, the invention provides sustainedrelease formulations of DNP including a therapeutically effective amountof DNP or a pharmaceutically acceptable salt or solvate thereof. As usedherein, “sustained release” or “extended release” refers to the factthat the DNP or pharmaceutically acceptable salt thereof is releasedfrom the formulation at a controlled rate so that therapeuticallybeneficial blood levels (which are below toxic levels) of the DNP orpharmaceutically acceptable salt thereof are maintained over an extendedperiod of time. Alternatively, “sustained release” or “extended release”means that the desired pharmacologic effect is maintained over anextended period of time.

In one aspect, the invention provides the potential for enhanced patientconvenience by reducing the frequency of dosing. In another aspect, thelower dosing frequency provides reduced side effects because the patientis exposed to lower peak concentrations of drug over time.

In certain embodiments, the invention provides an oral sustained releaseformulation. In yet other embodiments, the composition of the inventionis formulated for oral sustained release. However, the invention shouldnot be construed to be limited to only oral formulations, but ratherencompass any form of formulations that provides a low dose andsustained release of DNP.

In certain embodiments, the sustained release formulations of theinvention provide a controlled release of the drug over a longer periodthan observed for injectable or immediate release oral formulations(e.g., at least about 8-12 hours). In other embodiments, the sustainedrelease is over a period of time that may be as long as a month or moreand should be a release, which is longer that the same amount of agentadministered in bolus form. In yet other embodiments, the period of timeis greater than about one day, about two days, about one week, about twoweeks, about one month, about two months, and any and all ranges therebetween. In yet other embodiments, the period of time is between about12 and about 24 hours. In yet other embodiments, the period of time isabout 12 hours. In yet other embodiments, the period of time is about 14hours. In yet other embodiments, the period of time is about 24 hours.

In certain embodiments, the sustained release formulation isadministered once a day. In other embodiments, the sustained releaseformulation is administered twice a day. In yet other embodiments, thesustained release formulation is administered three times a day.

In certain embodiments, the sustained release provides a steady stateplasma concentration of a protonophore of the invention in a subject. Inother embodiments, the steady state plasma concentration ranges betweenabout 0.05 μM and about 200 μM. In yet other embodiments, the steadystate plasma concentration ranges between about 0.05 μM and about 50 μM.In yet other embodiments, the steady state plasma concentration rangesbetween about 0.5 μM and about 50 μM. In yet other embodiments, the peakplasma concentration is equal to or lower than about 400 μM. In yetother embodiments, the peak plasma concentration is equal to or lowerthan about 300 μM. In yet other embodiments, the peak plasmaconcentration is equal to or lower than about 30 μM. In yet otherembodiments, the steady state plasma concentration ranges between about1 μM and about 10 μM. In yet other embodiments, the steady state plasmaconcentration ranges between about 1 μM and about 5 μM. In yet otherembodiments, the steady state plasma concentration is about 5 μM. In yetother embodiments, the steady state plasma concentration is about 3 μM.

In certain embodiments, the sustained release formulation provides atissue concentration of the compound of the invention in a subject. Inother embodiments, the tissue concentration is equal to or lower than 10μM. In yet other embodiments, the tissue concentration is equal to orlower than 5 μM. In yet other embodiments, the tissue concentration isequal to or lower than 2 μM.

The compound of the invention or a salt thereof may be homogeneouslydispersed in the sustained release delivery system. In certainembodiments, the compound is present in the composition in an amount ofabout 1 mg to about 200 mg; about 1 mg to about 150 mg; about 1 mg toabout 125 mg; or about 1 mg to about 100 mg. In certain embodiments, thecompound or pharmaceutically acceptable salt thereof is present in thecomposition in an amount of about 2 mg to about 5 mg; about 5 mg toabout 80 mg; about 10 mg to about 70 mg; about 15 mg to about 60 mg;about 40 mg to about 80 mg; about 50 mg to about 70 mg; or about 45 mgto about 60 mg. In certain embodiments, the compound or pharmaceuticallyacceptable salt thereof is present in the composition in an amount ofabout 2 mg; about 20 mg, about 40 mg, about 60 mg, about 75 mg, about 80mg, about 100 mg, about 120 mg, about 140 mg, about 150 mg, about 160mg, about 175 mg, about 180 mg or about 200 mg.

In certain embodiments, the ratio of the compound or pharmaceuticallyacceptable salt thereof to the sustained release delivery system in thecomposition is generally from about 4:1 to about 1:25. In someembodiments, the ratio of the compound or pharmaceutically acceptablesalt thereof to the sustained release delivery system is generally fromabout 2.5:1 to about 1:4. In some embodiments, the ratio of the compoundor pharmaceutically acceptable salt thereof to the sustained releasedelivery system is generally from about 5:1 to about 1:5, about 4:1 toabout 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, about 1:1 toabout 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:1 toabout 1.2, and about 1:2 to about 1:3. In some embodiments, the ratio ofthe compound or pharmaceutically acceptable salt thereof to thesustained release delivery system is about 1:1, about 1:2, about 1:2.5,about 1:3, about 1:4, or about 1:5.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material that provides sustained releaseproperties to the compound. As such, the compounds may be administeredin the form of microparticles, for example, by injection or in the formof wafers or discs by implantation.

In certain embodiments, the compounds of the invention are administeredto a patient, alone or in combination with another pharmaceutical agent,using a sustained release formulation.

In certain embodiments, the sustained release formulation of a compoundof the invention is an orally administrable solid dosage formulation.Non-limiting examples of oral solid dosage formulations include tablets,capsules including a plurality of granules, sublingual tablets, powders,granules, syrups, and buccal dosage forms. In certain embodiments,tablets have an enteric coating or a hydrophilic coating.

In certain embodiments, the sustained release delivery system isprepared by dry granulation or wet granulation, before a compound of theinvention or pharmaceutically acceptable salt thereof is added, althoughthe components may be held together by an agglomeration technique toproduce an acceptable product. In the wet granulation technique, thecomponents (e.g., hydrophilic compounds, cross-linking agents,pharmaceutical diluents, cationic cross-linking compounds, hydrophobicpolymers, etc.) are mixed together and then moistened with one or moreliquids (e.g., water, propylene glycol, glycerol, alcohol) to produce amoistened mass that is subsequently dried. The dried mass is then milledwith conventional equipment into granules of the sustained releasedelivery system. Thereafter, the sustained release delivery system ismixed in the desired amounts with a compound of the invention or thepharmaceutically acceptable salt thereof and, optionally, one or morewetting agents, one or more lubricants, one or more buffering agents,one or more coloring agents, one or more second hydrophilic compounds,or other conventional ingredients, to produce a granulated composition.The sustained release delivery system and a compound of the inventionmay be blended with, for example, a high shear mixer. The compound ofthe invention is preferably finely and homogeneously dispersed in thesustained release delivery system. The granulated composition, in anamount sufficient to make a uniform batch of tablets, is subjected totableting in a conventional production scale tableting machine attypical compression pressures, i.e., about 2,000-16,000 psi. In certainembodiments, the mixture should not be compressed to a point where thereis subsequent difficulty with hydration upon exposure to liquids.

In some embodiments, a composition of the invention is prepared by drygranulation or wet granulation. The components of the sustained releasedelivery system are added, along with a compound of the invention orpharmaceutically acceptable salt thereof. Alternatively, all of thecomponents may be held together by an agglomeration technique to producean acceptable product. In the wet granulation technique, a compound ofthe invention or pharmaceutically salt thereof and the components (e.g.,hydrophilic compounds, cross-linking agents, pharmaceutical diluents,cationic cross-linking compounds, hydrophobic polymers, etc.) are mixedtogether and then moistened with one or more liquids (e.g., water,propylene glycol, glycerol, alcohol) to produce a moistened mass that issubsequently dried. The dried mass is then milled with conventionalequipment into granules. Optionally, one or more wetting agents, one ormore lubricants, one or more buffering agents, one or more coloringagents, one or more second hydrophilic compounds, or other conventionalingredients, are also added to the granulation. The granulatedcomposition, in an amount sufficient to make a uniform batch of tablets,is subjected to tableting in a conventional production scale tabletingmachine at typical compression pressures, i.e., about 2,000-16,000 psi.In certain embodiments, the mixture should not be compressed to a pointwhere there is subsequent difficulty with hydration upon exposure toliquids.

In certain embodiments, the average particle size of the granulatedcomposition is from about 50 μm to about 400 μm. In certain embodiments,the average particle size is from about 185 μm to about 265 μm. Theaverage density of the granulated composition is from about 0.3 g/mL toabout 0.8 g/mL. In certain embodiments, the average density is fromabout 0.5 g/mL to about 0.7 g/mL. The tablets formed from thegranulations are generally from about 4 Kp to about 22 kP hardness. Theaverage flow of the granulations is from about 25 to about 40 g/sec.

In one aspect, the invention provides a multilayer solid dosage form, inwhich the layers are formulated to release a compound of the inventionat different rates. For example, In certain embodiments, the secondlayer is an extended release layer that includes a compound of theinvention or a pharmaceutically acceptable salt thereof and a sustainedrelease delivery system designed to release a compound of the inventionor the pharmaceutically acceptable salt thereof at a controlled rate sothat therapeutically beneficial blood levels are maintained over anextended period of time (e.g., from about 8 to about 12 hours). Thefirst layer is an immediate release layer that includes a formulation ofa compound of the invention or a pharmaceutically acceptable saltthereof designed to release the compound of the invention or thepharmaceutically acceptable salt thereof at a rate that is faster thanthe rate of the second layer to achieve a therapeutically beneficialblood level in an immediate period of time (e.g., from about 1 to about2 hours). In some embodiments, the first layer includes a sustainedrelease delivery system. In some embodiments, the first layer does notinclude a sustained release delivery system.

In some embodiments, the weight ratio of the second layer to the firstlayer is about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 toabout 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 toabout 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 toabout 1:2. In certain embodiments, the weight ratio of the second layerto the first layer is about 5:1 to about 1:5. In yet other embodiments,the weight ratio of the second layer to the first layer is about 1:1 toabout 1:2. In yet another embodiments, the weight ratio of the secondlayer to the first layer is about 1:1, about 1:1.2, about 1:1.4, about1:1.6, about 1:1.8, or about 1:2. In certain embodiments, the weightratio of the second layer to the first layer is about 1:2. In certainembodiments, the weight ratio of the second layer to the first layer isabout 1:1.4. In some embodiments, the weight ratio of the second layerto the first layer is about 3:1, about 2.5:1, about 2:1, about 1.5:1. Incertain embodiments, the weight ratio of the second layer to the firstlayer is about 2.5:1.

In some embodiments, the multilayer dosage form further includes apharmaceutical disintegrant. The disintegrant promotes the dissolutionand absorption of a compound of the invention or pharmaceuticallyacceptable salt thereof from the immediate release layer. Non-limitingexamples of pharmaceutical disintegrants include croscarmellose sodium,starch glycolate, crospovidone, and unmodified starch. In certainembodiments, the disintegrant is in the first layer (i.e., the immediaterelease layer), of the dosage form. In certain embodiments, thedisintegrant is present in the layer in an amount of about 1.5 mg toabout 4.5 mg. In certain embodiments, the disintegrant is present in thelayer in an amount of about 2-10% by weight. In certain embodiments, thedisintegrant is present in the layer in an amount of about 5% by weight.When the layer contains a sustained release delivery system, the weightratio of the sustained release delivery system to the disintegrant is ina range of about 5:1 to about 1:5. In some embodiments, the ratio of thesustained release delivery system to the disintegrant is in a range ofabout 1:1 to about 3:1. In other embodiments, the ratio of the sustainedrelease delivery system to the disintegrant is in a range of about 2:1.

In some embodiments, the multilayer tablets of the invention areprepared by first preparing the immediate release layer and extendedrelease layer blends separately. The extended release layer is preparedas described elsewhere herein. The wet granulation of the extendedrelease layer is then dried and milled to an appropriate size. Magnesiumstearate is added and mixed with the milled granulation. The immediaterelease layer of the invention is prepared by first mixing a compound ofthe invention or the pharmaceutically acceptable salt thereof with oneor more diluents (e.g., microcrystalline cellulose). This mix is thenoptionally mixed with one or more disintegrants. The blend is mixed withmagnesium stearate. Finally, the immediate release layer blend and theextended release layer blend are compressed into multi-layer (e.g.,bi-layer) tablets.

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, delayed release and pulsatile releaseformulations.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Methods

The invention includes a method of preventing or treating a disease ordisorder in a subject in need thereof. The method comprisesadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising a compound selected from the groupconsisting of 2,4-dinitrophenol (DNP), a salt thereof, a solvatethereof, and any combinations thereof, wherein the composition providesa sustained release of the compound in the subject, whereby the diseaseor disorder in the subject is treated or prevented in the subject.

The invention further includes a method of increasing energy expenditurein a subject in need thereof. The method comprises administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound selected from the group consisting ofDNP, a salt thereof, a solvate thereof, and any combinations thereof,wherein the composition provides a sustained release of the compound inthe subject, whereby energy expenditure in the subject is increased.

In certain embodiments, the disease or disorder is at least one selectedfrom the group consisting of non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), hepatic steatosis, type 2 diabetes(T2D), acquired lipodystrophy, lipodystrophy (inherited), partiallipodystrophy, hypertriglyceridemia, obesity, metabolic syndrome, Rett'ssyndrome, metabolic syndrome associated with aging, metabolic diseasesassociated with increased reactive oxygen species (ROS), Friedreich'sataxia, insulin resistance, hepatic fibrosis, liver cirrhosis andhepatocellular carcinoma.

In certain embodiments, the subject is afflicted with at least onedisease or disorder selected from the group consisting of non-alcoholicfatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH),hepatic steatosis, type 2 diabetes (T2D), acquired lipodystrophy,lipodystrophy (inherited), partial lipodystrophy, hypertriglyceridemia,obesity, metabolic syndrome, Rett's syndrome, metabolic syndromeassociated with aging, metabolic diseases associated with increasedreactive oxygen species (ROS), Friedreich's ataxia, insulin resistance,hepatic fibrosis, liver cirrhosis and hepatocellular carcinoma.

In certain embodiments, the therapeutically effective dose of thecompound ranges from about 1 mg/kg/day to about 10 mg/kg/day. In otherembodiments, administration of the composition affords a steady stateplasma concentration of the compound ranging from about 0.05 μM to about200 μM in the subject. In yet other embodiments, administration of thecomposition affords a steady state plasma concentration of the compoundranging from about 0.5 μM to about 50 μM in the subject. In yet otherembodiments, administration of the composition affords a steady stateplasma concentration of the compound ranging from about 1 μM to about 10μM in the subject. In yet other embodiments, administration of thecomposition affords a steady state plasma concentration of the compoundin the subject ranging from about 3 μM to about 5 μM in the subject.

In certain embodiments, the steady state plasma concentration of thecompound in the subject is about 50 to about 100 times lower than thetoxic concentration of the compound in the subject.

In certain embodiments, administration of the composition affordstherapeutically effective levels of the compound in the subject for aperiod of time ranging from about 12 hours to about 24 hours. In certainembodiments, the composition is administered once, twice or three timesa day to the subject.

In certain embodiments, administration of the composition does not causesignificant systemic toxicity or significant increase in bodytemperature in the subject. In other embodiments, the significantsystemic toxicity is indicated by increase in levels of liver enzymes,blood urea nitrogen or creatinine as compared to the correspondinglevels in the subject in the absence of administration of thecomposition. In yet other embodiments, the composition is formulated fororal administration.

In certain embodiments, the method further comprises administering tothe subject at least one additional therapeutic agent. In otherembodiments, the composition and the at least one additional therapeuticagent are co-administered to the subject. In yet other embodiments, thecomposition and the at least one additional therapeutic agent areco-formulated.

In certain embodiments, the subject is a mammal. In other embodiments,the mammal is human.

Combination Therapies

The compounds useful within the methods of the invention may be used incombination with one or more additional compounds useful for treating adisease or disorder. These additional compounds may comprise compoundsthat are commercially available or synthetically accessible to thoseskilled in the art. These additional compounds are known to treat,prevent, or reduce the symptoms of a disease or disorder.

In non-limiting examples, the compounds useful within the invention maybe used in combination with one or more of the following therapeutics:

Pulmonary Hypertension Drugs: ambrisentan, bosentan, treprostinil,sildenafil, epoprostenol, treprostenol, iloprost, aldosterone receptorantagonists like spironolactone and eplerenone, angiotensin-convertingenzyme inhibitors such as trandolapril, fosinopril, enalapril,captopril, ramipril, moexipril, lisinopril, quinapril, benazepril, andperindopril;

Angiotensin II Inhibitors: eprosartan, olmesmian, telmismian, losartan,valsmian, candesartan, and irbesmian, anti-anginal agents likenitroglycerin, isosorbide mononitrate, and isosorbide dinitrate,anti-arrhythmic agents including moricizine, quinidine, disopyramide,phenyloin, propafenone, flecamide, mexilitene, lidocaine, procainamide,propranolol, acebutolol, amiodarone, dofetilide, dronedarone, sotalol,ibutilide, diltiazem, verapamil, nifedipine, nimodipine, felodipine,nicardipine, clevidipine, isradipine, bepridil, nisoldipine, adenosine,and digoxin;

β-adrenergic Receptor Antagonists: betaxolol, bisoprolol, metoprolol,atenolol, nebivolol, nadolol, carvedilol, labetalol, timolol, carteolol,penbutolol, pindolol, and esmolol;

Anti-Diabetic Agents: insulin, GLP-1 agonists, DPP4 inhibitors, SGLT-2inhibitors, secretagogues such as sulfonylurea, tolbutamide,acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide,glimepiride, glibenclamide, gliclazide, meglitinide such as nateglinide,senaglinide, repaglinide, insulin sensitizers such as biguanides,metformin, thiazolidinediones such as rosiglitazone, isaglitazone,darglitazone, englitazone, and pioglitazone;

α-Glucosidase Inhibitors: miglitol, voglibose, emiglitate, and acarbose;

Glucagon-Like Peptide Analogs and Agonists: exenatide, liraglutide, andtaspglutide, dipeptidyl peptidase-4 inhibitors like vildagliptin,sitagliptin, and saxagliptin;

Amylin Analogs: pramlintide;

Ligands or Agonists of Peroxisome Proliferator Activated Receptor(PPAR)-α, β, δ, and γ Cholesterol-Lowering Agents:hydroxymethylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors likestatins, such as, e.g., atorvastatin, fluvastatin, lovastatin,pitavastatin, pravastatin, rosuvastatin, and simvastatin;

Agonists of Retinoid X Receptors (RXR): ALRT-268, LG-1268, or LG-1069;glucokinase activators, inhibitors of hepatic enzymes involved instimulation of gluconeogenesis and/or glycogenolysis,

Diuretics: acetazolamide, dichlorphenamide, methazolamide, torsemide,furosemide, bumetanide, ethacrynic acid, amiloride, triamterene,indapamide, metolazone, methylclothiazide, hydrochlorothiazide,chlorothiazide, metolazone, bendroflumethiazide, polythiazide, andchlorthalidone;

Vasodilators: alprostadil, hydralazine, minoxidil, nesiritide, andnitroprus side;

Anti-Lipidemic Agents: cholestyramine, colestipol, clofibrate,gemfibrozil, probucol or dextrothyroxine;

Adipocytokines: leptin, adiponectin, and metreleptin;

Drugs for the treatment of Hyperlipidemia: fibrates, omega fatty acids,fish oil, statins.

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol.114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22:27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectivedose of a composition of the invention. The therapeutic dose of acomposition of the invention may be administered to the subject eitherprior to or after the onset of a disease or disorder. Further, severaldivided dosages, as well as staggered dosages may be administered dailyor sequentially, or the dose may be continuously infused, or may be abolus injection. Further, dosing of a composition of the invention maybe proportionally increased or decreased as indicated by the exigenciesof the therapeutic or prophylactic situation.

Administration of a composition of the invention to a patient,preferably a mammal, more preferably a human, may be carried out usingknown procedures, at dosages and for periods of time effective to treata disease or disorder in the patient. An effective amount of acomposition of the invention necessary to achieve a therapeutic effectmay vary according to factors such as the state of the disease ordisorder in the patient; the age, sex, and weight of the patient; andthe ability of the therapeutic formulation to treat a disease ordisorder in the patient. Dosing of a composition of the invention may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. A non-limiting example of an effective dose range for atherapeutic compound of the invention is from about 1 and 5,000 mg/kg ofbody weight/per day. One of ordinary skill in the art would be able tostudy the relevant factors and make the determination regarding theeffective amount of a composition of the invention without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount of acomposition of the invention. For example, the physician or veterinariancould start doses of a composition of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate acomposition of the invention in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thepatients to be treated; each unit containing a predetermined quantity oftherapeutic compound calculated to produce the desired therapeuticeffect in association with the required pharmaceutical vehicle. Thedosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the therapeutic compoundand the particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding/formulating such atherapeutic compound for the treatment of a disease or disorder in apatient.

In certain embodiments, a composition of the invention is formulatedusing one or more pharmaceutically acceptable excipients or carriers. Incertain embodiments, the formulations of the invention comprise acomposition of the invention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

In certain embodiments, a composition of the invention is administeredto the patient in dosages that range from one to five times per day ormore. In yet other embodiments, the formulations of the invention areadministered to the patient in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It is readily apparent to oneskilled in the art that the frequency of administration of the variouscombination compositions of the invention varies from individual toindividual depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any patient is determined by the attending physicaltaking all other factors about the patient into account.

In certain embodiments, the present invention is directed to a packagedpharmaceutical formulation comprising a container holding atherapeutically effective amount of a formulation of the invention,alone or in combination with a second pharmaceutical agent; andinstructions for using the compound to treat, prevent, or reduce one ormore symptoms of a disease or disorder in a patient.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, a composition of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of a disease or disorder. Using awax/pH-sensitive polymer mix, a gastric insoluble composition may beobtained in which the active ingredient is entrapped, ensuring itsdelayed release.

Parenteral Administration

For parenteral administration, a composition of the invention may beformulated for injection or infusion, for example, intravenous,intramuscular or subcutaneous injection or infusion, or foradministration in a bolus dose and/or continuous infusion. Suspensions,solutions or emulsions in an oily or aqueous vehicle, optionallycontaining other formulatory agents such as suspending, stabilizingand/or dispersing agents may be used.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Dosing

The dosing of a composition of the invention depends on the age, sex andweight of the patient, the current medical condition of the patient andthe progression of a disease or disorder in the patient being treated.The skilled artisan is able to determine appropriate dosages dependingon these and other factors.

A suitable dose of a compound of the present invention may be in therange of from about 0.001 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, dosing of a composition of theinvention may be initiated on Monday with a first subsequent low doseadministered on Wednesday, a second subsequent low dose per dayadministered on Friday, and so on. In certain embodiments, the compoundis dosed at least once a day. In yet other embodiments, the compound isdosed at least twice a day.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancelow dose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of theviral load, to a level at which the improved disease is retained. Incertain embodiments, patients require intermittent treatment on along-term basis upon any recurrence of symptoms and/or infection.

The compounds for use in the method of the invention may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. The data obtained from cell cultureassays and animal studies are optionally used in formulating a range ofdosage for use in human. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withminimal toxicity. The dosage optionally varies within this rangedepending upon the dosage form employed and the route of administrationutilized.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Animals:

Sprague-Dawley rats (300-400 g) and Zucker Diabetic Fatty rats (250 g)were ordered from Charles River Laboratories and allowed to acclimatefor at least one week before use. If not otherwise specified, rats werefed regular chow. In the NAFLD reversal model, rats were fed a saffloweroil based high fat diet (60% calories from fat) (Harlan) for the timeperiods specified, and were given free access to water at all times.

To determine whether ERDNP prevents NAFLD, rats were fed safflower oilhigh fat diet for two weeks while treated daily with ERDNP (1 mg/kg) orvehicle. To induce NASH, rats were fed methionine/choline deficient diet(Harlan) for eight weeks, then continued on this diet while treated themwith 1 mg/kg ERDNP or vehicle.

Surgery was performed under isoflurane anesthesia to place polyethylenecatheters (Instech Solomon) in the common carotid artery, jugular vein,and, where specified, antrum of the stomach (PESO, PE90, and PE90tubing, respectively). Rats were fasted for 6 hrs for measurement of DAGconcentrations, and overnight (16 hours) for all other studies. Forterminal studies, rats were euthanized by intravenous pentobarbital.

For the intragastric DNP infusion studies, rats previously fed high fatdiet for 2 weeks were placed in a Covance infusion harness attached to asingle-axis counter-balanced swivel mount and a stainless steelone-channel swivel (all from Instech Solomon) to protect the cathetersand allow the animals free access to the cage. DNP (2 mg/kg per day) orvehicle (10% dimethylsulfoxide/90% saline vehicle) was infusedcontinuously through the arterial line for 5 days.

ERDNP was formulated by Emerson Resources Inc. (Malvem, Pa.) withsustained-release ethylcellulose coating. Rats used for the NAFLDreversal studies were fed high fat diet for 2 weeks, at the end of whichthey were treated for three days with peanut butter to acclimate them tothis food. They were then treated daily with ERDNP mixed in peanutbutter, or peanut butter vehicle, at the doses specified in the textdaily for 5 days. Zucker Diabetic Fatty rats were treated with ERDNP (1mg/kg) in peanut butter or peanut butter vehicle daily for 14 days.Blood glucose was measured by glucometer (Abbott) from the tail veinevery three days.

Rats used for the NASH reversal studies were fed methionine/cholinedeficient diet for eight weeks as described above, then treated with 1mg/kg ERDNP or vehicle daily for six weeks while continuing onmethionine/choline deficient diet.

Normal male C57BL/6J mice were ordered at 12 weeks of age from Jacksonand were fed regular chow. After acclimating for five days, theyunderwent metabolic cage (CLAMS) analysis. The reported food intake doesnot include caloric intake from the small amount of peanut butter (˜250mg) used to administer ERDNP or vehicle; however, the peanut butterquantity was matched between groups.

Toxicity Studies:

Alanine aminotransferase, aspartate aminotransferase, and blood ureanitrogen were measured using the COBAS Mira Plus, and creatinine byLC/MS/MS. Body temperature was measured using a rectal probe (PhysitempInstruments). Histology slides were stained with hematoxylin & eosin,Sirius Red, and TUNEL stains.

Studies of Basal Metabolism:

Plasma insulin and glucagon were measured by radioimmunoassay. Plasmaglucose was measured enzymatically by the YSI Life Sciences 2700 SelectBiochemistry Analyzer. Plasma concentrations of twelve inflammatorymarkers, adiponectin, and FGF-21 were assessed by ELISA (QIAGEN, LifeTechnologies, and Millipore, respectively). Liver glycogen was measuredby amyloglucosidase digestion (Passonneau & Lauderdale, 1974, Anal.Biochem. 60:405-12). Non-esterified fatty acid concentrations weremeasured by an enzymatic kit (Wako).

To measure total glutamate enrichment from livers of rats infused with[3-¹³C] lactate, ˜100 mg frozen liver were homogenized in 400 μLice-cold 50% acetonitrile. The samples were centrifuged at 10,000 g for10 min, and the supernatant was isolated. After overnight storage at 4°C., the samples were centrifuged at 10,000 g for 10 min through aNanosep 100 k Omega filter (Pall Life Sciences). The flow-through wasseparated on a hypercarb column (Thermo Scientific; 4.6×100 mm; 5 μmparticle size) before ionization for multiple reaction monitoringanalysis by LC/MS/MS (Applied Biosystems MDS SCIEX, 4000 Q-TRAP).

To measure positional glutamate and alanine enrichment, the liversamples were extracted for nuclear magnetic resonance (NMR)spectroscopy. ˜4-6 g ground liver were centrifuged in ˜30 mL 7%perchloric acid. The pH of the supernatant was adjusted to 6.5-7.5 using30% potassium hydroxide and 7% perchloric acid as needed, and theextract was dehydrated by lyophilizing for 2-3 days. The extract wasresuspended in 500 μL potassium phosphate buffer: 2.4 mM NaCOOH, 30 mMK₂HPO₄, 10 mM KH₂PO₄, 20 mM DMSO (internal standard) in 100% D₂O. ¹³CNMR spectra were collected using the AVANCE 500-MHz NMR spectrometer(Bruker Instruments).

Spectra were acquired with relaxation time=1 s, dummy scans=32, andnumber of scans=8,192 per block×3 blocks. Correction factors fordifferences in T₁ relaxation times were determined from fully relaxedspectra of natural abundance glutamate and glucose solutions. The totalglutamate enrichment by LC/MS/MS was divided algebraically according tothe peak areas of [¹³C] glutamate for each carbon corrected for T1relaxation times.

Total glucose enrichment in the NMR extract was determined byderivatizing 20 μL of the NMR extract with 100 μL methanol, then dryingovernight in a Speed-Vac. The extract was then resuspended in 75 μL 1:1acetic anhydride:pyridine and heated for 20 min at 65° C. The sample wascooled and quenched with 25 μL methanol. The total m+1 glucoseenrichment of each sample was measured by gas chromatography/massspectrometry using previously described methods (Shulman et al., 1985,J. Clin. Invest. 76:757-764). m+2, m+3, m+4, m+5, and m+6 enrichmentwere found to be negligible (<5% of m+1 enrichment at steady-state). ¹³CNMR spectra were used to determine relative concentrations of [¹³C]glucose. As for glutamate, the total glucose enrichment by massspectrometry was divided algebraically to measure the enrichment at eachglucose carbon.

To measure positional alanine enrichment from [3-¹³C] lactate infusedrat livers, liver samples were extracted for NMR as described above, andthe enrichment at [2-¹³C] and [3-¹³C] alanine was measured byproton-observed, carbon-edited NMR as we have reported (Alves et al.,2011, Hepatology 53:1175-1181).

Lipid Measurements:

Plasma triglycerides were measured using a Wako reagent. Liver andquadriceps triacylglycerol were extracted using the method of Bligh andDyer (Bligh & Dyer, 1959, Can. J. Biochem. Physiol. 37:911-7) andquantified spectrophotometrically using a reagent from DiagnosticChemicals Ltd. Liver and quadriceps DAG and ceramide concentrations weremeasured by liquid chromatography/mass spectrometry/mass spectrometry(LC/MS/MS) (Yu, et al., 2002, J. Biol. Chem. 277:50230-6), as wereacylcarnitine concentrations (An, et al., 2004, Nat. Med. 10:268-74).Very low-density lipoprotein (VLDL) export was measured (Lee, et al.,2011, Hepatology 54:1650-60). Liver acetyl and malonyl CoAconcentrations were measured by LC/MS/MS (Hosokawa, et al., 1986, Anal.Biochem. 153:45-9; Roughan, 1994, Biochem. J. 300:355-8).

Markers of Liver Fibrosis and Apoptosis:

Caspase-3 and -9, smooth muscle actin, and inflammatory cytokines weremeasured in liver homogenates by ELISA (MyBioSource, NeoBioLab,MyBioSource, and SABiosciences, respectively), and were normalized tototal protein content measured by Bradford assay. Liver hydroxyprolinecontent was measured (Cai, et al., 2014, J. Pharmacol. Exp. Therap.349:94-98). Collagen mRNA was measured in liver, and UCP1 mRNA in BAT byqPCR (Kumashiro, et al., 2011, Proc. Natl. Acad. Sci. USA108:16381-16385).

Glucose Tolerance Tests:

Rats were given an intraperitoneal bolus of 50% dextrose (1 mg/kg) attime zero. Plasma was obtained by drawing blood through the venouscatheter at times 0, 5, 10, 20, 30, 45, 60, and 90 min and centrifugedimmediately. Plasma glucose and insulin concentrations were measured asdescribed elsewhere herein.

Measurement of Insulin Sensitivity and Hepatic Fluxes:

Basal and insulin-stimulated glucose turnover were measured using asteady-state (120 min) infusion of [6,6]²H₂ glucose (Erion, et al.,2013, Endocrinology 154:36-44).

Muscle and BAT glucose uptake were measured by injection of[¹⁴C]2-deoxyglucose into the venous line (Samuel, et al., 2007, J. Clin.Invest. 117:739-45).

PKCε and θ translocation in liver and quadriceps, respectively, weremeasured by Western blot (Choi, et al., 2007, Proc. Natl. Acad. Sci. USA104:16480-5).

Liver PC flux (V_(pc)), flux through the TCA cycle (V_(tca)), andsubstrate contributions to the TCA cycle (V_(pdh), V_(fa)) were measuredby steady-state [3-¹³C] lactate infusion (Perry, et al., 2013, Cell.Metab. 18:740-8).

Measurement of Gluconeogenic Protein Concentrations:

Protein concentrations of pyruvate carboxylase, glucose-6-phosphatase,and fructose-1,6-bisphosphatase were measured using antibodies fromSanta Cruz Biotechnology (Kumashiro, et al., 2013, Diabetes 62:2183-94).CD69 was measured using an antibody from Novus Biologicals (Kumashiro,etb al., 2013, Diabetes 62:2183-2194). PKC translocation was measured(Samuel, et al., 2007, J. Clin. Inv. 117:739-745).

DNP and ERDNP Kinetics Studies:

Rats consumed the concentrations of DNP or ERDNP specified in the textorally, mixed in peanut butter. All rats used for the kinetics studiesconsumed the entirety of the peanut butter and protonophore within 2minutes.

DNP concentrations were measured in plasma, liver, kidney, WAT,quadriceps, heart, and brain of Sprague-Dawley rats by LC/MS/MS (Perry,et al., 2013, Cell. Metab. 18:740-8). Rats treated with DNP weresacrificed 1 hour after treatment with DNP, and those treated with ERDNPwere sacrificed 8 hours after treatment, as these were the timesdetermined in the plasma DNP studies to represent the peak plasma DNPconcentrations (FIGS. 21A-21B). The minimal detection limit of themethod was determined to be 0.05 μM, as this was the minimumconcentration of solutions of known quantities of DNP in DMSO measuredby the method with less than 20% error in each of three replicates.

Statistical Analysis:

Differences were assessed by the 2-tailed unpaired Student's t-test whentwo groups were compared, or by ANOVA with Bonferroni's multiplecomparisons test when three groups were compared. P-values <0.05 wereconsidered significant. Data are reported as mean±S.E.M.

Histology Studies:

Liver and kidney samples were prepared and stained with hematoxylin &eosin by the Yale Research Histology core, and analyzed as described(Kleiner et al., 2005, Hepatology 41:1313-1321).

Measurement of Liver Glycogen Content:

Hepatic glycogen content was assessed by amyloglucosidase digestionusing previously described methods (Passonneau and Lauderdale, 1974,Anal. Biochem. 60:405-412).

Assessment of Potential Gluconeogenic Markers:

Plasma concentrations of twelve inflammatory markers were measured byELISA (QIAGEN, Valencia, Calif.). Adiponectin was measured by ELISA bythe Yale Diabetes Research Center Physiology Core. Lactate was measuredby COBAS, and FGF-21 by ELISA (Millipore).

Measurement of Plasma and Tissue DNP Concentrations:

LC/MS/MS method development and analysis were performed on the AppliedBiosystems 4000 QTRAP (Foster City, Calif.), equipped with a Shimadzuultra fast liquid chromatography (UFLC) system. Electrospray ionization(ESI) source with negative-ion detection was shown as the most sensitivefor both qualitative and quantitative analysis of DNP. The quantitativeanalysis of DNP was monitored in MRM mode with an ion pair(183.0/109.0). The preferred parameters are: curtain gas 25; collisiongas 9, probe temperature 480° C.; ion source gas 1 20; ion source gas 225; declustering potential (DP) −45 V; entrance potential (EP) −10 V;collision energy (CE) −35 V and collision cell exit potential −12 V.

Extraction from Plasma Samples for LC/MS/MS Analysis:

Plasma samples (10-100 μL) were mixed with 2.0 ml pre-chilledchloroform/methanol (v/v: 2/1) containing 0.01% BHT in 5 ml glass vials,to which was added 250 μl water together with 10 nmol DNP-D₃ and 10 nmolDNPME-D₆ as the internal standards. The mixtures were vortexed for 10seconds before centrifugation at 4000 rpm for 10 min. The bottom organiclayer was carefully collected and dried with a steady stream of nitrogengas. The residual was reconstituted in 200 μl methanol for LC/MS/MSanalysis for DNP and/or DNPME metabolites.

Extraction from Various Tissue Samples for LC/MS/MS Analysis:

Frozen tissue samples (˜100 mg) were weighed and suspended in 2 mlmicrocentrifuge tubes with 1.6 ml pre-chilled chloroform/methanol (v/v:2/1) containing 0.01% BHT and one metal bead, and then added 10 nmolDNP-d3 (Cambridge Isotopes, Andover, Mass.) as the internal standards.The tissue samples were disrupted with Qiagen TissueLyser at 30 Hz for15 min, and then transferred into 5 ml glass vials, followed by additionof 0.5 ml chloroform and 250 μl water into each samples. The sampleswere centrifuged at 4000 rpm for 10 min after vortexed for 10 seconds.The bottom organic layer was collected and dried with a gentle flow ofnitrogen. The residual was reconstituted in 200 μl methanol for LC/MS/MSanalysis of DNP.

Example 1 Formulation of DNP

DNP was formulated for extended release. In certain embodiments,extended release of DNP reduces toxicity and maximizes efficacy. Inother embodiments, the DNP formulation comprise a DNP extruded or druglayered bead that is coated with a sustained release coating in a dosagesize suitable to deliver 1.0 mg/kg of DNP over a 12-24 hour period.

In certain embodiments, the polymers used in the sustained releasecoating work independent of pH, as they are a combination of soluble andinsoluble polymers, allowing release through pores created by thesoluble polymer. In other embodiments, the formulated drug is absorbedin the acidic stomach and/or neutral intestine. In yet otherembodiments, a multiparticulate system allows for lower variance indissolution rates (as a coating failure in a tablet could result incomplete release of the dose whereas a coating failure in one of manyparticulates would result in a minimally altered dissolution profile).

In certain embodiments, a DNP bead is prepared using extrusion andspheronization. In order to achieve controlled release, two polymersystems were selected for evaluation: a EUDRAGIT® RS/RL system and ahydroxypropylcellulose (HPC)/ethylcellulose (EC) system. Initialformulation work focused on evaluating the HPC/EC and EUDRAGIT® systemsat various coating levels, and the HPC/EC system was selected forfurther investigation. Table 1 illustrates raw materials used forpreparation of ERDNP spheres.

TABLE 1 Generic Name Trade Name Supplier Mannitol MANNITOL 100 SDRoquette Microcrystalline cellulose MCC PH-101 FMC (MCC) BiopolymerHydroxypropylmethyl PHARMACOAT ® 606 Shin-Etsu cellulose (HPMC)Ethylcellulose (EC) ECN 10 Dow Hydroxypropylcellulose (HPC) KLUCEL ® EFAshland GMS Emulsion PLASACRYL ® T20 Emerson Polysorbate 80 — EvonikTalc — Brenntag Dibutyl sebacate (DBS) — Vertellus Distilled water —Emerson Ethanol (200 proof) — SpectrumUncoated 2,4-DNP Extruded Sphere

In certain embodiments, a desired dose weight is selected based on threefactors: development of a dosage weight that would feasibly deliver therequested dose (1.0-1.5 mg/kg) to subjects, avoidance of uniformityissues, and avoidance of issues with the sustained release coating.Based on the selected dose weight and API concentration, a formulationusing common excipients for extruded beads was developed. The initialbead formulation (NK15-142, CU03-170, CU03-171) was designed to deliver1.5 mg DNP/60 mg beads; however, this did not take into account a 22%moisture content in the active pharmaceutical ingredient (API), whichresulted in an actual API concentration closer to 1.17 mg/60 mg beads.Subsequent batches were formulated similarly to the latter but wereadjusted to deliver 1.0 mg DNP/50 mg beads (or 1.17 mg/58.5 mg beads).Table 3 illustrates the composition of the initial uncoated beadformulations.

TABLE 2 NK15-142, CU03-170, CU03-171 NK15-155 % g % % g % g w/w wet/ w/wg dry/ mg/ w/w wet/ w/w dry/ mg/ Ingredient (wet) batch (dry) batch dose(wet) batch (dry) batch dose Mannitol 65.3 391.8 65.7 391.8 39.4 65.4394.8 65.8 394.8 32.9 MCC 32.0 192.0 32.2 192.0 19.3 31.8 192.0 32.0192.0 16.0 HPMC 0.2 1.2 0.2 1.2 12.1 0.2 1.2 0.2 1.2 0.1 2,4-DNP¹ 2.515.0 2.0 11.7 1.2 2.5 15.4 2.0 12.0 1.0 Water — 200.0² — — — — 200.0 — —— Total 100.0 600.0 100.0 596.7 60.0 100.0 603.4 100.0 600.0 50.0¹2,4-DNP contains 22% moisture. The % w/w wet shows the weight percentof wet API dispensed for the batch. The dry basis % w/w and mg/dosetakes into account only the solids concentration, omitting the 22%moisture in the API. ²Water is used as a processing aid and does notappear in the final product. It is not represented in the % w/w or finalbatch weight and is only included to show the quantity added forprocessing.

To prepare the uncoated spheres, the solid components were added to ahigh shear mixer and mixed until visually uniform (1 minute at mix speed300 rpm). 200 ml water were added, and the solution was mixed untilvisually uniform (2-3 minutes at mix speed 300 rpm). The material wasdischarged and extruded with a 0.7 mm die face, 9 shims, at 90 rpm. Theextrudate was divided into two batches, and each was spheronized with a2 mm plate at 980 rpm for 2 minutes. The beads were then placed in anoven and dried until the final moisture content was <7.5% based onstarting weight.

Controlled Release Bead Formulation Development

In order to achieve controlled (12-24 hr) release, two polymer systemswere selected for evaluation: an EUDRAGIT® RS/RL system and ahydroxypropylcellulose (HPC)/ethylcellulose (EC) system.

The EUDRAGIT® system functions by combining permeable and impermeable pHindependent swelling polymers, allowing the drug to diffuse out overtime, controlled by both the ratio of permeable and impermeable polymerand the amount of polymer applied.

The HPC/EC system functions by combining soluble and insoluble polymersto create pores in the coating through which the drug diffuses overtime, also controlled by the ratio of the polymers and the amount ofpolymer applied. Formulations for the two coating suspensions aredescribed in Table 3.

TABLE 3 NK15-146 (Eudragit System) NK15-143 (HPC/EC System) % % gsolids/ % % g solids/ Ingredient w/w solids batch g/batch w/w solidsbatch g/batch EUDRAGIT ® 42.0 30.0 168.0 560.0 — — — — RS 30D EUDRAGIT ®10.5 30.0 42.0 140.0 — — — — RL 30D ECN 10 — — — — 6.92 100.0 103.8103.8 KLUCEL ® EF — — — — 0.77 100.0 11.5 11.5 PLASACRYL ® 7.8 20.0 20.8104.0 — — — — T20 TEC 2.4 100.0 32.0 32.0 — — — — Polysorbate 80 0.933.0 4.0 12.0 — — — — Talc — — — — 1.54 100.0 23.1 23.1 DBS — — — — 0.77100.0 11.5 11.5 DI Water 36.4 0.0 0.0 485.3 9.00 0.0 0.0 135.0 Ethanol —— — — 81.00 0.0 0.0 1215.0 Total 100.0 20.0 266.8 1333.3 100.00 10.0%100.0 1500.0

To generate the controlled-release coating with the EUDRAGIT® system,the Plasacryl was shaken and mixed. While mixing, the EUDRAGIT®, water,TEC, and PS80 were added. The suspension was then passed through a 30mesh screen and mixing was continued. To coat the DNP spheres producedas described elsewhere herein, the FLM1 fluid bed was set up with bottomspray Wurster coating with a 1.2 mm liquid nozzle, 3 mm air cap, Wurstercolumn, 400 mesh inlet screen, conidor plate, and 40 mesh filters. Thespheres were loaded into the fluid bed and coated using the parametersin Table 4. The beads were then dried for a minimum of 10 min with aninlet temperature of 40° C.

TABLE 4 Parameter Target range Spray rate 15-25 g/min Inlet temperature44-47° C. Product temperature 34-37° C. Nozzle air 40 psi Process air55-70 cfm

Two lots of beads coated with the HPC/EC system were coated and sampledat three theoretical weight gains (11.7%, 13.3%, and 15% for LotNK15-144 and 7.5%, 10%, and 11.7% for Lot NK15-157). The final coatedbead formulation for each lot (based on a 1 mg dose and the maximumcoating applied) is illustrated in Table 5.

TABLE 5 Lot # NK 15-144 NK 15-157 (15.0% weight gain) (11.7% weightgain) Ingredient % w/w mg/dose % w/w mg/dose 2,4-DNP drug spheres 87.051.3 89.5 50.0 ECN 10 9.0 5.3 7.3 4.1 KLUCEL ® EF 1.0 0.6 0.8 0.5 Talc2.0 1.2 1.6 0.9 Dibutyl sebacate 1.0 0.6 0.8 0.5 Total 100.0 58.9 100.055.9

Beads coated with the EUDRAGIT® system showed a tendency to block(clump) into the run. Extended release beads prepared with the HPC/ECsystem were evaluated for assay and dissolution. The results aredescribed in Table 6.

TABLE 6 Lot # % Label Claim NK15-144-01 (11.7%) 97.4 NK15-144-12 (13.3%)93.0 NK15-144-23 (15.0%) 94.4 NK15-157-01 (7.5%) 92.8 NK15-157-12(10.0%) 95.9 NK15-157-23 (11.7%) 96.7Exemplary ERDNP Formulation

A sustained release dosage form (beads) was developed using an HPC/ECcoating system. The 12 hour targeted release profile was confirmedthrough in vitro dissolution testing. The final formulation representingthe DNP drug spheres is illustrated in Table 7.

TABLE 7 Ingredient % w/w g/batch mg/dose Mannitol 65.8 394.8 32.9 MCC32.0 192.0 16.0 HPMC 0.2 1.2 0.1 2,4-DNP 2.0 12.0 1.0 Total 100.0 600.050.0These drug spheres were used to generate the controlled-release DNP drugwith composition as illustrated in Table 8.

TABLE 8 Ingredient % w/w mg/dose 2,4-DNP drug spheres 89.5 50.0 ECN 107.3 4.1 KLUCEL ® EF 0.8 0.5 Talc 1.6 0.9 DBS 0.8 0.5 Total 100.0 55.9

Example 2 Continuous Low Dose Intragastric DNP Reduced Liver and MuscleLipid Content and Improved Whole Body Insulin Sensitivity

In order to test whether a continuous, low dose intragastric infusion ofDNP achieving sustained plasma DNP concentrations of ˜3 μM would lead toreductions in hepatic steatosis and improve whole body insulinsensitivity, rats were infused with intragastric DNP (2 mg/kg per day)for 5 days. This infusion rate of intragastric DNP resulted in steadystate plasma and tissue DNP concentrations of ˜3 μM and ˜1 μM in plasmaand liver respectively with negligible concentrations of DNP in skeletalmuscle (FIG. 2A). By comparison, a dose of DNP (5 mg/kg) in the ratresults in a peak plasma DNP concentration of ˜120 μM and peak DNP liverconcentrations of ˜60 μM.

These very low concentrations of DNP resulted in marked (>80%)reductions in plasma triglycerides as well as >50% reductions in liverand muscle TAG and diacylglycerol (DAG) content. These reductions inliver and muscle TAG/DAG content were associated with markedimprovements in whole body insulin sensitivity as indicated by lowerfasting plasma glucose and insulin concentrations (FIGS. 1, 14A-14F,14K-14L). In contrast to these marked improvements in whole body insulinsensitivity, there were no changes in tissue ceramide content and nosystemic toxicities associated with these chronic intragastric DNPinfusions as reflected by no observed changes in liver enzymes, bloodurea nitrogen (BUN) or creatinine (FIGS. 14F-14J).

Example 3 Safety and Efficacy of ERDNP

Since these continuous intragastric DNP infusion studies demonstratedthat sustained plasma and tissue concentrations of DNP in the 1-3 μMrange reversed hepatic steatosis and insulin resistance in a rat modelof NAFLD, safety and efficacy studies of an extended release DNP (ERDNP)formulation were performed, in which the formulation was fed to ratsmixed in small amounts (˜1 g) of peanut butter.

In contrast to DNP, which caused a dose dependent increase in bodytemperature and doses at and above 25 mg/kg, ERDNP had a negligibleeffect on body temperate, with only a detectable 0.5° C. increase at 100mg/kg (FIGS. 4A-4B).

To compare the safety and efficacy of ERDNP and DNP at reversingdiet-induced NAFLD, two-week high fat fed rats were treated with varyingdoses of ERDNP or DNP daily for five days. The lowest effective dose ofERDNP to decrease liver triglyceride was 0.5 mg/kg, whereas that of DNPwas 5 mg/kg (FIGS. 4C-4D). No changes to alanine aminotransferase (ALT),aspartate aminotransferase (AST), BUN or creatinine were observed withany of the doses of ERDNP, whereas daily DNP treatment at doses above 1mg/kg raised AST concentrations, doses above 2 mg/kg raised ALT, and thehighest dose of DNP raised BUN (FIGS. 15A-15H). Thus the ratio of toxic(100 mg/kg) to therapeutic dose (0.5 mg/kg) was 200 for ERDNP, ascompared to 0.4 (toxic dose 2 mg/kg, therapeutic dose 5 mg/kg) for DNP,yielding a 500 fold improvement in the toxic to effective dose ratio forERDNP compared to DNP.

Example 4 Pharmacokinetics of ERDNP and DNP

The improved safety to efficacy ratio of ERDNP relative to DNP wascorrelated with much lower plasma DNP concentrations with ERDNP comparedto DNP. The area under the plasma DNP concentration curve was 20-fold inrats treated with 1 mg/kg ERDNP (effective dose used in these studies)versus 25 mg/kg DNP (lowest dose that produced an increase in bodytemperature) (211±18 vs. 4,311±555, P=0.001; FIG. 5A), with a time ofmaximum concentration of DNP just one hour with DNP treatment, versus8-12 hours after treatment with ERDNP. The maximum plasma concentrationof DNP was 30-fold higher with DNP treatment than with ERDNP, but thehalf-life of ERDNP was twice as long (8-12 hours for DNP vs. 16-24 hoursfor ERDNP). As predicted by the plasma concentration data, tissue DNPconcentrations were 10-20-fold lower following both one and five days of1 mg/kg ERDNP treatment than in rats treated once with 25 mg/kg DNP(FIGS. 5B-5D). There were no differences in tissue DNP concentrations inrats treated for 6 weeks with DNP (FIG. 5E) compared to rats treated for1 or 5 days. As predicted by these data, tissue DNP concentrations 24hours after a dose of ERDNP were undetectable (FIG. 5F), as were tissueconcentrations of DNP at 48 and 72 hours after ERDNP treatment,demonstrating that DNP does not accumulate in tissues with chronic ERDNPtreatment.

Although peak plasma DNP concentrations following 1 mg/kg treatment werenot different between rats treated with DNP and ERDNP, the area underthe curve of DNP concentration was doubled with treatment with CRNDPrelative to DNP, likely accounting for ERDNP's improved efficacy atequimolar doses (FIGS. 4C-4D, 21A-21E). A strong correlation wasobserved between plasma DNP concentration and body temperature (FIG.21F). In certain embodiments, DNP toxicity is a function of the maximumplasma DNP concentration. Tissue concentrations of DNP following 1 mg/kgERDNP treatment (effective dose) were 25-100-fold lower than DNPconcentrations after the lowest toxic dose of DNP (25 mg/kg), withoutany change in the ratio of plasma/tissue DNP after plasma DNPconcentrations had plateaued (FIGS. 21G-21I). Tissue DNP concentrations24 hours after a dose of ERDNP were undetectable (below the lower limitof detection of the method, 0.05 μM) (FIG. 21H), as were tissueconcentrations of DNP 48 and 72 hours after ERDNP treatment,demonstrating that DNP does not accumulate in tissues with chronic ERDNPtreatment. Accordingly, DNP concentrations following chronic DNPtreatment did not differ significantly from tissue concentrations afterone ERDNP treatment, and were under 10 μM in all tissues (FIG. 21J).Tissue DNP concentrations correlated linearly with ERDNP dose other thanin brain where measured DNP concentrations were at the lower limit ofdetection of the method (0.05 μM) (FIG. 21K), implying the absence ofdose-dependent tissue DNP metabolism. In concert with this, plasma DNPconcentrations were not different after chronic (5 daily) doses of 1mg/kg ERDNP than after the first dose (FIG. 21L).

Six weeks of treatment with ERDNP at 1 mg/kg was similarly welltolerated and did not result in any alterations in behavior, foodintake, body weight, body temperature, elevations in liver enzymes(ALT/AST), BUN, or creatinine and there was no evidence of cellularinjury or necrosis in liver or kidney histology (FIGS. 16A-16G).

Tissue DNP concentrations following the last dose did not differ fromtissue DNP concentrations after one or five daily doses of ERDNP (FIG.16H). Without wishing to be limited by any theory, the toxicity of a DNPderivative can be predicted by the maximum concentration of DNP, whereasits efficacy can be predicted by the area under the curve of plasma DNPconcentrations.

Example 5 ERDNP Treatment Reduces Hypertriglyceridemia, HepaticSteatosis and Insulin Resistance

To examine whether ERDNP reduces tissue lipid content and improve wholebody insulin sensitivity, a well-established high fat fed rat model ofNAFLD and insulin resistance was treated with daily ERDNP (1 mg/kg) orvehicle for five days. Despite identical body weight at the time ofstudy, the ERDNP treated rats had an approximately 30-40% reduction infasting plasma glucose, fatty acid and triglyceride concentrations, a30% increase in high density lipoprotein concentration and a 50%reduction in plasma insulin concentrations (FIGS. 6A-6D, 17A-17B).

Rats treated with ERDNP manifested improved glucose tolerance, withlower plasma glucose and insulin concentrations during anintraperitoneal (IP) glucose tolerance test (FIGS. 6E-6F, 17G-17H).

In order to more fully assess the effect of ERDNP on whole body insulinsensitivity, hyperinsulinemic-euglycemic clamps combined withradiolabeled glucose were performed to assess insulin action in liverand skeletal muscle (FIGS. 18A-18B). Consistent with improved whole bodyinsulin sensitivity by IP glucose tolerance tests, the ERDNP-treatedrats required two-fold more glucose to maintain euglycemia during thehyperinsulinemic-euglycemic clamp study (FIGS. 7A, 18C). Thisimprovement in insulin-stimulated whole body glucose metabolism in theERDNP treated animals could be attributed to increases in both liver andmuscle insulin sensitivity as reflected by a 2.5-fold increase ininsulin-stimulated peripheral muscle glucose uptake (FIG. 7B) and athree-fold greater suppression of hepatic glucose production inERDNP-treated rats during the hyperinsulinemic-euglycemic clamp (FIGS.7C-7D).

There may be a strong causal relationship between diacylglycerol(DAG)-induced nPKC activation and insulin resistance in liver andskeletal muscle. Consistently, ERDNP-treated rats had lowertriacylglycerol (TAG) and DAG content and decreased protein kinase C(PKC)ε and PKCθ translocation in liver and skeletal muscle respectively(FIGS. 8A-8B, 18D-18G). As predicted by their lower fasting plasmaglucose concentrations, flux through pyruvate carboxylase in theERDNP-treated rats was 25% lower than controls, and this reduction in PCflux was associated with a 60% reduction in hepatic acetyl CoA, which isa key allosteric regulator of PC activity (FIGS. 8C-8D). In contrastthere were no differences in gluconeogenic protein expression in theERDNP-treated rats compared to the control animals (FIGS. 17C-17F).

Example 6 ERDNP Treatment Reduces Hepatic Pyruvate Carboxylase Flux andIncreases Rates of Hepatic Mitochondrial Fat Oxidation

The effects of ERDNP on rates of hepatic mitochondrial TCA (V_(TCA))flux, V_(PDH) flux, V_(fa) flux and V_(PC) flux were assessed using anovel combined NMR-LC/MS/MS method (Perry, et al., 2013, Cell. Metab.18:740-8). Consistent with their reduced liver lipid content, liver TCAcycle flux was increased 60% in ERDNP-treated rats after one day oftreatment. Furthermore this increase V_(TCA) flux was entirely due to a65-70% increase in rates of hepatic fat oxidation (FIG. 8E). Incontrast, there were no differences in fat oxidation relative to V_(TCA)in kidney, brain, heart, or skeletal muscle, indicating that theuncoupling effect of ERDNP is confined to the liver (FIG. 17I).

In one aspect, the lower skeletal muscle TAG content may be due, atleast in part, to reduced hepatic very low-density lipoprotein (VLDL)export as a result of increased hepatic fatty acid oxidation. Liver VLDLexport was reduced by 80% in ERDNP-treated animals (FIG. 8F).

In contrast, no difference was observed in liver or quadricepsacylcarnitines and ceramides, liver glycogen content, plasmaconcentrations of twelve inflammatory markers, adiponectin, or FGF-21,thus dissociating these metabolites and adipocytokines fromERDNP-induced improvement in liver and muscle insulin responsiveness(FIGS. 18H-18S). In addition, there were no differences in brown adiposetissue mass, insulin-stimulated glucose uptake, or uncoupling protein-1(UCP1) mRNA expression (FIGS. 18X-18Z).

To examine whether uncoupling with ERDNP reduces tissue lipid contentand improves insulin sensitivity, a well-established high fat fed ratmodel of NAFLD and insulin resistance was treated with daily ERDNP (1mg/kg) or vehicle for five days. Despite identical body weight and whiteadipose tissue content at the time of study, ERDNP treated rats werestrikingly more insulin sensitive than their vehicle-treatedcounterparts, manifesting 30-40% reductions in fasting plasma glucose,fatty acid and triglyceride concentrations, a 30% increase in highdensity lipoprotein concentration and a 50% reduction in plasma insulinconcentrations, without any difference in hepatic gluconeogenic proteinexpression (FIGS. 6A, 6C, 6G, 17A, 17C-17F, 17J-17K).

Example 7 ERDNP Improve Whole-Body Insulin Resistance

Rats treated with ERDNP manifested improved glucose tolerance, withlower plasma glucose and insulin concentrations throughout anintraperitoneal (IP) glucose tolerance test (FIGS. 6E-6F, 17I-17J). Inorder to more fully assess the effect of ERDNP on whole body insulinsensitivity, hyperinsulinemic-euglycemic clamps with radiolabeledglucose were performed to assess insulin action in liver and skeletalmuscle (FIGS. 18A-18B). Consistent with improved whole body insulinsensitivity, the ERDNP-treated rats required two-fold more glucose tomaintain euglycemia during the hyperinsulinemic-euglycemic clamp study(FIGS. 7A, 18C). Without wishing to be limited by any theory, thisimprovement in insulin-stimulated whole body glucose metabolism in theERDNP-treated animals can be attributed to increases in both liver andmuscle insulin sensitivity, as reflected by a 2.5-fold increase ininsulin-stimulated peripheral muscle glucose uptake and a 3-fold greatersuppression of hepatic glucose production in ERDNP-treated rats duringthe hyperinsulinemic-euglycemic clamp (FIGS. 7C, 18T). There is a strongcausal relationship between ectopic diacylglycerol (DAG) accumulationand insulin resistance in liver and skeletal muscle. ERDNP-treated ratshad lower triacylglycerol (TAG) and DAG content and decreased proteinkinase Cε (PKCε) and PKCθ translocation in liver and skeletal musclerespectively (FIGS. 8A-8B, 18D-18F, 18U-18V). The reduction in skeletalmuscle triglycerides were associated with 40% lower plasma triglycerideconcentrations and an 80% reduction in liver very low-densitylipoprotein (VLDL) export (FIG. 6B, 8F), explaining the reduced musclelipid content as a result of liver-specific uncoupling.

The data presented herein indicates absence of hyperthermia in the ratsand stand in contrast to the reduced UCP1 mRNA expression in micetreated with a far higher dose of DNP in drinking water (˜89mg/[kg-day]), emphasizing the safety of the present formulation. ERDNPwas similarly effective at preventing the development of NAFLD: rats fedhigh fat diet for 2 weeks and concurrently fed ERDNP had lower fastingplasma glucose, NEFA and insulin concentrations associated with 50-90%reductions in triglyceride concentrations in liver, plasma, and skeletalmuscle (FIGS. 22A-22F). In order to more conclusively examine the effectof ERDNP treatment on whole-body energy metabolism, Comprehensive LabAnimal Monitoring System (CLAMS) metabolic cage studies were performedin mice fed ERDNP or vehicle and no difference was observed in anyparameter examined (FIGS. 23A-23H). These data again show that very lowlevels of uncoupling confined to the liver, which cannot be measuredwith the relatively insensitive CLAMS studies, are sufficient to reduceliver fat content and improve whole-body insulin resistance, withoutaffecting food intake, behavior, or whole-body energy expenditure.

Example 8 ERDNP Treatment Reverses Diabetes in Zucker Diabetic FattyRats

Since ERDNP treatment safely reversed NAFLD, hypertriglyceridemia, andhepatic and peripheral insulin resistance in high fat fed rats. Incertain embodiments, ERDNP could reverse hyperglycemia,hypertriglyceridemia and hepartic steatosis in a well-established obeserat model of T2D, the Zucker Diabetic Fatty (ZDF) rat (a hyperphagicleptin-deficient obese rat model).

To this end, high fat fed ZDF rats were treated with ERDNP (1 mg/kg)daily for 14 days. ERDNP treatment was associated with a progressivereduction in plasma glucose concentrations and a 400 mg/dL decrease infasting plasma glucose concentrations after two weeks of treatment alongwith an 80% decrease in fasting plasma insulin concentrations despiteidentical body weight before and after treatment (FIGS. 9A-9B, 19A).Consistent with improved insulin sensitivity, ZDF rats also had lowerfasting plasma triglyceride concentrations after 14 days of ERDNPtreatment (FIGS. 8C-8D). ERDNP-treated rats also displayed a 60%reduction in hepatic acetyl CoA content, which IS a key regulator ofgluconeogenesis and glycemia in diabetic animals (FIG. 19L).

ERDNP-treated rats manifested improved glucose tolerance during theintraperitoneal glucose tolerance test, with 50-80% reductions in plasmaglucose and insulin concentrations at each time point during the GTT,and 60-70% reductions in total glucose and insulin area under the curvein the ERDNP-treated group (FIGS. 9E-9F, 19B-19C). These improvements ininsulin sensitivity and glucose tolerance were associated with 65% and55% reductions in liver and quadriceps TAG concentration, respectively(FIGS. 19D-19E). There was no detectable renal toxicity with thistwo-week treatment as reflected by no changes in plasma BUN, and amodest reduction in creatinine concentrations (FIGS. 19F-19I). Incontrast, liver enzymes (AST, ALT) and hepatic TAG content wereincreased in ZDF rats before treatment reflecting hepatic steatosisassociated with liver inflammation in these poorly controlled diabeticanimals; ERDNP treatment normalized these parameters reflecting reversalof hepatic steatosis and liver inflammation in this rat model of NASHand T2D with ERDNP treatment (FIGS. 19F-19I). Histologic analysisconfirmed the resolution of NAFLD with ERDNP treatment in this poorlycontrolled diabetic model (FIG. 9I), highlighting the possibility thatERDNP is a therapeutic agent for NAFLD-associated liver disease.

Example 9 ERDNP Treats NAFLD-Induced NASH

To investigate the possibility that ERDNP ameliorates or treatsNAFLD-induced NASH, rats were fed a methionine/choline deficient diet(MCD) for 8 weeks to induce NASH. Six weeks of ERDNP treatment reducedliver triglyceride concentrations by 90% and normalized plasmatransaminase concentrations (FIGS. 20A-20C). Consistent with thereduction in liver inflammation indicated by the normalization oftransaminase concentrations, ERDNP treated rats displayed lowerconcentrations of five inflammatory cytokines in the liver and reducedliver CD69 protein, a marker of activated T cells, in ERDNP treated ratsrelative to control rats (FIGS. 20D, 24A-24B). Histological analysisconfirmed the resolution of NAFLD and liver fibrosis in ERDNP treatedrat livers, with a 90% reduction in the liver fibrosis score andaccompanying reductions in collagen mRNA, smooth muscle actin protein,and hydroxyproline concentrations (FIGS. 20E-20I). Rats treated withERDNP also exhibited reductions in apoptosis, with lower caspase 3 andcaspase 9 protein expression, but no detectable difference in TUNELstaining (FIGS. 20J-20K, 24C). Patients with hepatic cirrhosis manifestreduced postprandial hepatic glycogen synthesis, and thus hepaticglycogen content was measured in MCD fed rat livers. An 80% increase inhepatic glycogen synthesis was observed in ERDNP-treated rats associatedwith reversal of fasting hypoglycemia in these animals (FIG. 24D-24E).ERDNP improved liver synthetic function, as indicated by 20% increasesin plasma albumin concentrations (FIG. 20L). These data demonstrateimprovements in hepatic protein and carbohydrate synthetic function, inaddition to reversal of liver fibrosis, in a NASH model and emphasizethe efficacy of ERDNP as a therapeutic agent for NAFLD-associated NASHto prevent liver cirrhosis and potentially hepatocellular carcinoma.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of treating a disease or disorder in asubject in need thereof, the method comprising orally administering tothe subject a therapeutically effective amount of a pharmaceuticalcomposition comprising: at least one pharmaceutically acceptable carrierand a therapeutically effective amount of 2,4-dinitrophenol (DNP) or asalt or solvate thereof; wherein the DNP or a salt or solvate thereof,is coated with an extended release coating comprising: i) at least onewater soluble polymer; and ii) at least one water insoluble polymer,wherein the therapeutically effective amount of DNP is about 1 to about10 mg/kg/day or about 1 to about 200 mg; wherein administration of thecomposition to a subject provides a steady state plasma concentration ofDNP ranging from about 0.05 μM to about 200 μM in the subject, andwherein the composition does not cause significant systemic toxicity orsignificant increase in body temperature in the subject and, wherein thedisease or disorder is at least one selected from the group consistingof nonalcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH), hepatic steatosis, type 2 diabetes (T2D),acquired lipodystrophy, inherited lipodystrophy, partial lipodystrophy,hypertriglyceridemia, obesity, metabolic syndrome, Rett's syndrome,metabolic syndrome associated with aging, metabolic diseases associatedwith increased reactive oxygen species (ROS), Friedreich's ataxia,insulin resistance, hepatic fibrosis, liver cirrhosis and hepatocellularcarcinoma, whereby the disease or disorder in the subject is treated inthe subject.
 2. The method of claim 1, wherein the therapeuticallyeffective dose of the DNP ranges from about 1 mg/kg/day to about 10mg/kg/day.
 3. The method of claim 1, wherein administration of thecomposition provides a steady state plasma concentration of the DNPranging from about 0.5 μM to about 50 μM in the subject.
 4. The methodof claim 3, wherein administration of the composition provides a steadystate plasma concentration of the DNP ranging from about 3 μM to about 5μM in the subject.
 5. The method of claim 2, wherein the steady stateplasma concentration of the DNP in the subject is about 50 to about 100times lower than the toxic concentration of the compound in the subject.6. The method of claim 1, wherein administration of the compositionprovides therapeutically effective levels of the DNP in the subject fora period of time ranging from about 12 hours to about 24 hours.
 7. Themethod of claim 1, wherein the composition is administered once, twiceor three times a day to the subject.
 8. The method of claim 1, whereinthe significant systemic toxicity is indicated by increase in levels ofliver enzymes, blood urea nitrogen or creatinine as compared to thecorresponding levels in the subject in the absence of administration ofthe composition.
 9. The method of claim 1, wherein the composition isformulated for oral administration.
 10. The method of claim 1, furthercomprising administering to the subject at least one additionaltherapeutic agent.
 11. The method of claim 10, wherein the compositionand the at least one additional therapeutic agent are co-administered tothe subject.
 12. The method of claim 11, wherein the composition and theat least one additional therapeutic agent are co-formulated.
 13. Themethod of claim 1, wherein the subject is a mammal.
 14. The method ofclaim 13, wherein the mammal is human.
 15. A method of increasing energyexpenditure in a subject in need thereof, the method comprising orallyadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising; at least one pharmaceuticallyacceptable carrier and a therapeutically effective amount of2.4-dinitrophenol (DNP) or a salt or solvate thereof; wherein the DNP ora salt or solvate thereof, is coated with an extended release coatingcomprising: i) at least one water soluble polymer: and ii) at least onewater insoluble polymer, wherein the therapeutically effective amount ofDNP is about 1 to about 10 mg/kg/day or about 1 to about 200 mg; whereinadministration of the composition to a subject provides a steady stateplasma concentration of DNP ranging from about 0.05 μM to about 200 μMin the subject, and wherein the composition does not cause significantsystemic toxicity or significant increase in body temperature in thesubject and, wherein the subject is afflicted with at least one diseaseor disorder selected from the group consisting of nonalcoholic fattyliver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepaticsteatosis, type 2 diabetes (T2D), acquired lipodystrophy, inheritedlipodystrophy, partial lipodystrophy, hypertriglyceridemia, obesity,metabolic syndrome, Rett's syndrome, metabolic syndrome associated withaging, metabolic diseases associated with increased reactive oxygenspecies (ROS), Friedreich's ataxia, insulin resistance, hepaticfibrosis, liver cirrhosis and hepatocellular carcinoma, whereby energyexpenditure in the subject is increased.
 16. The method of claim 15,wherein the therapeutically effective dose of the DNP ranges from about1 mg/kg/day to about 10 mg/kg/day.
 17. The method of claim 15, whereinadministration of the composition provides a steady state plasmaconcentration of the DNP ranging from about 0.5 μM to about 50 μM in thesubject.
 18. The method of claim 17, wherein administration of thecomposition provides a steady state plasma concentration of the DNPranging from about 3 μM to about 5 μM in the subject.
 19. The method ofclaim 15, wherein administration of the composition providestherapeutically effective levels of the DNP in the subject for a periodof time ranging from about 12 hours to about 24 hours.
 20. The method ofclaim 15, wherein the composition is administered once, twice or threetimes a day to the subject.
 21. The method of claim 15, wherein thesignificant systemic toxicity is indicated by increase in levels ofliver enzymes, blood urea nitrogen or creatinine as compared to thecorresponding levels in the subject in the absence of administration ofthe composition.
 22. The method of claim 15, wherein the composition isformulated for oral administration.
 23. The method of claim 15, furthercomprising administering to the subject at least one additionaltherapeutic agent.
 24. The method of claim 15, wherein the subject is amammal.
 25. The method of claim 24, wherein the subject is a human. 26.An oral pharmaceutical composition comprising: at least onepharmaceutically acceptable carrier and a therapeutically effectiveamount of 2,4-dinitrophenol (DNP), or a salt or solvate thereof; whereinthe DNP, or a salt or solvate thereof, is coated with an extendedrelease coating comprising: i) at least one water soluble polymer; andii) at least one water insoluble polymer, wherein the therapeuticallyeffective amount of DNP is about 1 to about 10 mg/kg/day or about 1 toabout 200 mg; wherein administration of the composition to a subjectprovides a steady state plasma concentration of DNP ranging from about0.05 μM to about 200 μM in the subject, and wherein the composition doesnot cause significant systemic toxicity or significant increase in bodytemperature in the subject.
 27. The pharmaceutical composition of claim26, wherein the composition does not change alanine aminotransferase(ALT) levels in the subject after administration.
 28. The pharmaceuticalcomposition of claim 26, wherein the composition does not changeaspartate aminotransferase (AST) levels in the subject afteradministration.
 29. The pharmaceutical composition of claim 26, whereinthe steady state plasma concentration of the DNP in the subject is about50 to about 100 times lower than the toxic concentration of the DNP inthe subject.
 30. The pharmaceutical composition of claim 26, whereinadministration of the amount of the composition affords therapeuticallyeffective levels of the DNP in the subject for a period of time rangingfrom about 12 hours to about 24 hours.
 31. The pharmaceuticalcomposition of claim 26, wherein the significant systemic toxicity isindicated by increase in levels of liver enzymes, blood urea nitrogen orcreatinine, as compared to the corresponding levels in the subject inthe absence of administration of the composition.
 32. The pharmaceuticalcomposition of claim 26, wherein the composition further comprises atleast one additional therapeutic agent.
 33. The pharmaceuticalcomposition of claim 26, wherein the at least one water soluble polymercomprises hydroxypropylcellulose.
 34. The pharmaceutical composition ofclaim 26, wherein the coated DNP is in a bead or sphere form.
 35. Thepharmaceutical composition of claim 26, wherein the at least one waterinsoluble polymer comprises ethylcellulose.
 36. The pharmaceuticalcomposition of claim 26, wherein the subject is a mammal.
 37. Thepharmaceutical composition of claim 36, wherein the mammal is human.